CN1226925A - Phosphate starvation-inducible proteins - Google Patents

Abstract

This invention provides proteins, especially protein kinases and glucosidases, and phosphate transproton which are expressed under conditions of phosphate deprivation. Further provided are nucleic acids and nucleic acid constructs encoding these proteins, cells containing the nucleic acids described and transgenic photosynthetic organisms with altered phosphate-inducible enzyme activity.

Description

The induced protein of phosphate starvation

Correlation technique of the present invention

Phosphorus is to one of of paramount importance nutritive ingredient of plant.It is that plant-growth is necessary, and is nucleic acid, phosphatide, metabolic intermediate and numerous other structural composition of biomolecules.

In plant, only can absorbed exogenous phosphorus be inorganic phosphorus (Pi) (Bieleski, 1973).In the time of phosphatic quantity not sufficient, plant just can't grow and output healthy and strongly.If lack phosphorus, growth just stops, and plant can be dead.

Because plant is a sessile organism, they must deal with the biochemical pressure of environment, for example, and extreme temperature, the lacking and arid etc. of nutrient.This photosynthetic organism set or finite motion to other is all the same.Therefore, plant and other photosynthetic organism all need the signal conduction pathway to start the poor environment stimulated cells is replied.

For a long time, people have known is the influence (Salisbury and Ross, 1985) that the interim feature of blooming and quantitative characteristic all are subjected to the ratio of the level of phosphoric acid salt and nitrogen in the plant.Phosphatic relative content height can cause the maturation of plant, and the phosphoric acid salt of low relatively amount can cause the generation of not blooming.What also known is that phosphatic level can influence the partition rate of biomass between root and limb.Specifically, the phosphatic preferred growth (Lefebvre et al., 1982) that causes root that lacks back.Therefore, in a lot of environment, the phosphatic main limiting factor of utilizing degree just to become limiting plant growth and photosynthetic organism breeding.

The essence of replying for the phosphate starvation of plant has had numerous people to study.But, however, still know little for the absorption and the metabolic molecular mechanism of regulation and control phosphorus.In general, for the interference of environment, replying of plant is that significant form and physiological change take place.

People are once to determining that institute's inductive protein has carried out numerous trials under the phosphate starvation condition.People such as Fife (1990) have carried out in the research of the intravital protein labeling of black mustard (Brassicanigra) cell, and cell wherein is grown in rich phosphorus or the low-phosphorous substratum.Adopt two-dimensional gel electrophoresis, they have proved under the situation that Pi lacks and have produced four kinds of protein, and produce a kind of protein in the sound cell of nutrition.Other some researchs had reported once that the shortage of Pi can improve proteinic synthetic and soluble protein (the Hawkesfordand Belcher in tomato root cells culture of serous coat, 1991), and strengthen in the tomato suspension cell six kinds of proteinic secretions (Goldstein et al., 1989).Also proved with the black mustard suspension cell of the proteinic gene of beta-glucosidase enzyme homologous under the Pi starvation conditions in induced and be high level (Malboobi and Lefebvre, 1995).

As the ADP of adenosine nucleoside acid, cellular energy material and the part of ATP, phosphorus is vital to bio-energy kinetics.And then the adding of the covalency phosphate group of biological substrate and removal (being respectively phosphorylation and dephosphorylation) play " ON/OFF " effect of a kind of modulability in cellular metabolism and the signal conduction of being everlasting.For example, the phosphorylation and the dephosphorylation of some membrane-bound receptor protein kinases and its substrate are the keys of multiple signal conduction pathway, the passage that comprises plant hormone such as ethene (Kieber et al., 1993) and dormin (Anderberg and Walker-Simmons, 1992).With activity (Tantikanjana et al., 1993 of being pollinated and being fertilized self uncompatibility relevant and also relating to by the coded protein kinase of S-locus gene; Zhang and Walker, 1993).

Lack the knowledge of the expression under the environment for the protein of absorption that influences phosphorus and accumulation and at phosphoric acid salt, be used for the phosphate metabolism of the growth of photosynthetic organism of commercial and industrial target and breeding and regulation and control for understanding and all be basic property.And then, for the synthetic of these proteinic genes of coding with determine, can develop the transgenosis photosynthetic organism that to be used for various uses.

The present invention's general introduction

The invention provides by modified plant or other photosynthetic organism one or more protein expression and/or the active method that changes phosphorus metabolism replying of lacking of phosphorus.The present invention also further provides and has made plant and other photosynthetic organism more effectively utilize phosphorus to carry out metabolic method.Compound of the present invention provides the method that changes phytomorph by the modification phosphorus metabolism.In some applications, these modifications will be limited in seed, and the compound that this anti-trophicity phosphorus of phytinic acid wherein stores will be lowered.

The invention still further relates to for phosphorus lack institute's inductive the phosphorus of plant and photosynthetic organism absorb and metabolism in related protein (psr protein) carry out complementary DNA, the recombinant DNA structure of coded DNA (gene), these genes and contain the carrier of these protein DNAs of coding or complementary DNA, comprise that the integral body of these materials or its are a part of.

Specifically, the invention provides the DNA of protein kinase, beta-glucosidase enzyme and the phosphoric acid salt transporton of coding black mustard and Arabidopis thaliana, it is transcribed and can be induced by phosphate starvation, and the RNA that is so transcribed is provided.Nucleic acid of the present invention (comprising DNA and RNA) encoded protein matter is that to be different from other known protein matter kinase whose, because it has the part in unique aminoacid sequence.Beta-glucosidase enzyme of the present invention and phosphoric acid salt transporton are different from known beta-glucosidase enzyme and phosphoric acid salt transporton on sequence, the difference because their expression level depends on phosphatic disappearance.

Other nucleic acid of the present invention comprises nucleic acid or its part with the nucleic acid array complementation of black mustard and Arabidopis thaliana (Arabidopsisthaliana); Relevant but distinguishing with the nucleotide sequence of black mustard and Arabidopis thaliana and can be under phosphoric acid salt shortage condition inductive nucleic acid; And owing to sudden change, substitute, the different adorned analogue of the nucleotide sequence with black mustard and Arabidopis thaliana that disappearance etc. causes.Containing 20 of above-mentioned nucleic acid or the primer and the probe of more a plurality of continuous nucleotides also all is a part of the present invention.The present invention also comprises proteinic homologue of psr and protein intimate with it.

Therefore, one of nucleic acid of the present invention is antisense oligonucleotide, the oligonucleotide of triple helices form, or other can suppress oligonucleotide by the psr protein expression of nucleic acid encoding as herein described.Such oligonucleotide can seal these protein expressions by several approach, for example, formation with triple helices prevents transcribing of psr protein coding gene, or combines in any mode that is combined into functional protein that prevents with mRNA by genetic transcription.Usually, according to the mode of effect, oligonucleotide of the present invention comprises about 20 sequences to about 200 or more a plurality of Nucleotide, and its nucleotide sequence with psr protein coding gene or the mRNA that transcribes is identical or complementary.

The present invention also comprises but nucleic acid of the present invention is connected to come the included nucleic acid of production the present invention with the expression vector of regulating sequence, plasmid or reorganization with mode of operation.In a preferred embodiment, but the expression vector of reorganization comprise and regulate the nucleic acid that the sequence place of working is connected, and be applicable to conversion to vegetable cell.

The present invention comprises that also the psr of the present invention that expresses one or more is proteinic by cell that transform or genetically modified.In a preferred embodiment, described transgenic cell is a vegetable cell.The transgenic plant that the present invention includes expression proteinic nucleic acid of psr of the present invention or carrier and produce.The present invention also further comprises the part of transgenic plant, comprises the seed, tissue culture and the protista that are produced by nucleic acid or carrier of the present invention.

The present invention also provides the expression vector of the reorganization that is used for transformed plant cells, but it comprises and regulates the dna molecular that the sequence place of working is connected, to express nucleotide sequence is the RNA molecule of antisense, and nucleotide sequence wherein has and the basic homologous sequence of nucleotide sequence by SEQ ID NO:1, SEQID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11-17 or SEQ ID NO:19-27 representative.

The present invention further comprises the psr method of protein that utilizes nucleic acid of the present invention to prepare to have the psr protein active.This method is included in cell transformed or the transgenic cell of cultivating the expression vector that contains reorganization in the suitable culture base, but described carrier comprises nucleic acid of the present invention and the adjusting sequence that is connected with its place of working, then, separates psr protein.The present invention also provides separated psr protein or has had the psr protein active and polypeptide that have homology with the represented aminoacid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:18 or its part.

The present invention also comprises and described new psr protein (or one partial sequence) bonded antibody and antibody fragment.In a preferred embodiment, antibody is monoclonal antibody.

The present invention also further comprises the method for the psr protein expression that reduces photosynthetic organism, preferred plant, the step of this method comprises separated nucleic acid is incorporated in the biology, wherein said nucleic acid is the antisense thing that has with the basic homologous nucleic acid of nucleotide sequence of coding psr proteinic gene, and described nucleotide sequence is SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11-17 or SEQ ID NO:19-27.

Method of the present invention can be used to overwhelm dominant allele, and its expression rate is lowered, under extreme case, and the phenotype that can " eliminate allelotrope ".When have in the cell to mRNA for part or such result can take place during the complementary sequence completely.What it is generally acknowledged is, when having sense-rna to exist, and mRNA: reduced widely but the result of the generation of the hybrid of antisense thing is the detection level of target gene product.Antisense is transcribed and can be caused that stable state has the reduction of adopted mRNA level, and its reason may be to put upside down to be enhanced, or specific duplex is attacked [Murray and Crockett (1992)] by double-stranded RNase.Must consider the structure of inverted defined gene, expression level must be sufficiently high, and want temporary transient and express synchronous with target gene.In addition, for fear of the expression of influence, must guarantee the specificity of sequence in the site of unique function.Can reach such effect by the segmental selection of coding psr nucleic acid sequences to proteins to the zone of translating or do not translate.

The present invention also further provides the method that reduces plant psr protein expression level, the nucleic acid with separated that this method comprises is incorporated into the step in the plant, wherein said nucleic acid causes the common inhibition to gene, and these genes are identical with the psr nucleic acid sequences to proteins or basic homologous.

The present invention further provides the method that the psr activity of proteins to plant reduces or raises, comprise separated nucleic acid is incorporated into step in the plant, wherein said nucleic acid causes the psr proteinic production that has been changed, and make it more effective, or to (natural generation) functional psr protein itself so that the mode that its activity is lowered is gone functionalization or disturbed.

Therefore, the invention provides and regulate photosynthetic organism the phosphatic method of replying of its ambient level, and the mechanism of modifying these biological phosphate metabolisms.The mode in the phosphoric acid salt path of this modified plant has numerous advantages that are better than the traditional plant method of cultivation, the most important thing is, these modifications can promptly be finished, and specific characteristic can be modified, even can to introduce new be not the characteristic of the part of Plant Genome.

Brief description of drawings

Fig. 1 be different Pi handle to the black mustard cell in 7 days growth endogenous phosphate content (middle null symbol) and the effect of dry-matter accumulation (filled symbols).Initial p i concentration in substratum be zero (zero, ●), 1.25mM (, ■) and 10mM (△, ▲).

Fig. 2 is poly-(A) that extracts in 7 days the black mustard suspension cell of growth ⁺The external translation product of RNA ³⁵The densitometric scan that the radioactive automatic developing of the SDS-polyacrylamide gel of S-mark is taken a picture, substratum is the MS substratum, does not contain Pi (dotted line) or contains 1.25mM Pi (broken broken line) or 10mM Pi (solid line).The polymer weight zone of A group expression gel; Molecular weight region in the expression of B group; C group expression lower molecular weight zone.The arrow indication is corresponding to the peak value of inductive polypeptide.Provided the molecular weight of estimating on the X-axis.

Fig. 3 is a column diagram, is illustrated in the difference of the relative populations of the expressed mRNA of black mustard suspension cell that the Pi of different concns cultivates down.The length of each post is represented the zone under its pairing peak value, therefore, promptly represents the relative richness of this mRNA.The label at each peak is seen Fig. 2.Provided the molecular weight of estimating on the Y-axis.

Fig. 4 has shown the Northern blot of total RNA, extracts from (a) of 7 ages in days a small amount of Pi and handles, and (b) has added 1.25mM Pi and (c) has added the black mustard suspension cell of 10mM Pi.Tub A is with the alpha-tubulin that compares.The data on each group left side are the estimated values to the mRNA size of cloning corresponding to each psr.Each band contains the total RNA of 30 μ g.

What Fig. 5 showed is the dna sequence dna (SEQ ID NO:1) of inducing the protein kinase psrPK (psr1) of generation from Arabidopis thaliana under phosphate starvation, and the aminoacid sequence of coded protein kinase (SEQ ID NO:2).

Fig. 6 is that coding is induced the cDNA sequence (SEQ ID NO:1) of the protein kinase psrPK (psr1) of generation and the comparison of the coded cDNA sequence (SEQ ID NO:3) of the homologous protein kinase that comes from black mustard from Arabidopis thaliana under phosphate starvation.Residue in the picture frame is represented two same sections between the sequence.

Fig. 7 code displaying is induced the protein kinase psrPK (psr1) of generation from Arabidopis thaliana under phosphate starvation dna sequence dna (SEQ ID NO:1), underscore is capitalized and added to its 3 ' terminal sequence.

Fig. 8 is the comparison of aminoacid sequence (SEQ ID NO:4) with the aminoacid sequence of other protein kinase of the aminoacid sequence (SEQ ID NO:2) of Arabidopis thaliana psrPK (psr1) and black mustard.

Fig. 9 shows the Computer Analysis of the aminoacid sequence that Arabidopis thaliana psrPK protein is inferred.

Figure 10 shows the result of experiment of carrying out according to 7 pairs of nuclears of embodiment.

Figure 11 is the comparison of 3 ' terminal of the 3 ' terminal of cDNA sequence of the 3 ' terminal of eDNA sequence of Arabidopis thaliana psrPK (psr1) and black mustard and other cDNA sequence.

Figure 12 A-12D has represented to have justice and antisense psrPK (psr1) structure, wherein component (CaMV-35S) or seed-specific (Arabin-pro) promotor and justice (psr1) is arranged or antisense (the psrPK gene fusion of α-psr1).

Figure 13 is Arabidopis thaliana clone's (being determined by the dna probe of restriction enzyme enzymolysis and the Southern blotting of black mustard psr3.1cDNA) a collection of illustrative plates, and the position of psr3.2 is pointed out by arrow.

Figure 14 A and Figure 14 B show phosphate starvation induction beta-glucosidase enzyme (psr3.2) dna sequence dna (SEQ ID NO:5) with and the aminoacid sequence (SEQ IDNO:6) of inferring.

Figure 15 is black mustard psr3.1 cDNA clone's (psr3.1B) the nucleotide sequence (SEQID NO:7) and the aminoacid sequence (SEQ ID NO:8) of its deduction.

Figure 16 is Arabidopis thaliana psr3.1cDNA clone's (psr3.1A) the nucleotide sequence (SEQ ID NO:9) and the aminoacid sequence (SEQ ID NO:10) of its deduction.

Figure 17 A and Figure 17 B show Arabidopis thaliana psr3.2, the comparison of the aminoacid sequence of the aminoacid sequence of psr3.1A and black mustard psr3.1B and other plant beta-glucosidase enzyme.

Figure 18 is the partial dna sequence (SEQ ID NO:11 and SEQID NO:12) of the psr2 of black mustard.

Figure 19 is the partial dna sequence (SEQ ID NO:13 and SEQID NO:14) of the psr4 of black mustard.

Figure 20 is the partial dna sequence (SEQ ID NO:15 and SEQID NO:16) of the psr5 of black mustard.

Figure 21 is the partial dna sequence (SEQ ID NO:17 and SEQID NO:18) of the psr6 of black mustard.

Figure 22 is the partial dna sequence (SEQ ID NO:19 and SEQID NO:20) of the psr7 of black mustard.

Figure 23 is the partial dna sequence (SEQ ID NO:21) of the psr8 of black mustard.

Figure 24 is the partial dna sequence (SEQ ID NO:22 and SEQID NO:23) of the psr9 of black mustard.

Figure 25 is the partial dna sequence (SEQ ID NO:24 and SEQ ID NO:25) of the psr10 of black mustard.

Figure 26 is the partial dna sequence (SEQ ID NO:26 and SEQ ID NO:27) of the psr11 of black mustard.

Figure 27 A-27B shows that the Southern Blot of Arabidopsis genomic dna analyzes.

Figure 28 is the conversion of Arabidopis thaliana and the collection of illustrative plates that produces the offspring.

Detailed description of the present invention

The present invention relates to the method, particularly plant of new generation photosynthetic organism, and the biology that produces like this, the use of such method. It is to induce in the photosynthetic organism cell of phosphate starvation that the present invention is based in part on the transcript and expression of finding some protein. Therefore, the invention provides the separated DNA of at least one functional part of the protein (psr protein) of encoded light synthesising biological, wherein, phosphatic shortage is induced transcribing of this DNA in cell. Specifically, the three class psr protein of encoding, namely the gene of ser/thr (serine/threonine) protein kinase, β-glucosyl enzym and phosphate transporton has been separated and serializing. As shown in drawings, the nucleic acid of all coding psr polypeptide, and the homologue of these psr nucleic acid all comprises in the present invention.

Separate the clone

In investigation black mustard suspension cell in the initial stage step of replying of Pi hunger, mRNA Pi is hungry and that provide the cell of Pi to extract is external to translate whether the protein that compares to study the black mustard cell is synthetic can be regulated and control on the level of translating. At first, the black mustard suspension cell was grown 7 days in the culture medium that contains 1.25mM Pi, made all cells be in identical metabolism state. Then with cell transfer in the culture medium with different Pi initial concentrations. To next 7 days growth conditions be serious Pi disappearance (0, Pi), moderate Pi disappearance (1.25mM Pi) or be rich in Pi (10mM Pi) (Lefebvre et al., 1990). Under moderate Pi disappearance, time the 2nd day, plant cell has absorbed whole Pi, and is being rich under the condition of Pi, cultivates after 7 days, and plant does not absorb whole Pi yet. Separate total mRNA from each culture, the material that these are separated is translated external. Resulting polypeptide separates with high-resolution SDS-polyacrylamide gel.

Phosphate starvation does not cause the synthetic total change (Fife et al., 1990) of protein of black mustard. But, comparing with the cell that has added 10mM Pi, the inventor isolates a small amount of RNA constantly from the cell that disappearance Pi processes, show that the protein synthetic ratio may be lowered. This and the high-load free amino acid of in cell, observing match (Duff et al., 1994).

The densitometric scan of SDS-polyacrylamide gel has been determined four peptide species (be approximately 31.7,32.3,52.5 and 64.8kDa), and they only exist only in the sample of Pi hunger (seeing Fig. 2). The body internal protein synthesis analysis to the black mustard suspension cell that the people (1990) such as this and Fife report matches. Adopt two-dimensional gel electrophoresis, they have shown new synthetic four kinds of protein (64kDa, pI5.2 under the Pi deletion condition; 41 kDa, pI5.6; 27kDa, pI5.7; 27kDa, pI5.2), and in the sufficient cell of nutrition, produce a kind of protein (33kDa, pI5.1). This relatively is best supposition, because the modification after translating is merely able to carry out in living cells, the derivable true protein of being reported by people such as Fife has the size identical with the protein that detects in this research.

Set up cDNA library from the mRNA of the black mustard cell separation of several disappearances. With this cDNA library of different screening by hybridization, the cDNA probe of use process from disappearance Pi with the black mustard cell that has added 10mM Pi (nutrition is abundant) prepare. Identified the mRAN that several representatives are preferentially translated under the Pi deletion condition. These phosphate starvation responsiveness (psr) clones (121 clones) are put into 11 different homology groups, and its homology is determined by crisscrossing. Northern blot shows that the expression of each gene of these 11 groups is controllable on the level of translating (Malboobi and Lefebrve, 1995). Northern blot shows, all is derivable under the corresponding gene Pi starvation conditions hundred moderates and serious; That is, having got rid of extreme environment pressure causes cell death to the side effect of inducing of these genes.

As shown in Figure 3, the expression of some gene (grey bar) in the interpolation of 7 ages in days that experienced moderate Pi disappearance also be derivable in the cell of 1.25mM Pi. The size of corresponding protein is about 31.7,32.3,52.5 and 64.8kDa. If the external rate of translating is independent of courier's kind, then the from the beginning expression to four kinds of distinguishing effable couriers is just higher comparatively speaking in the cell that disappearance Pi processes, and with those the abundantest mRNA in these cells Comparatively speaking this be. According to the form of expressing, the protein of these gene codes is played the part of respectively active metabolism and structural role at cell in to the adaptation of Pi pressure. The evaluation of the derivable gene of phosphate and characterization: protein kinase psr gene

To the serializing of DNA and Analysis and Identification subsequently in the gene, i.e. psrPK (psr1), as protein kinase (SEQ ID NO:3), its expression is derivable in the black mustard cell of phosphate starvation. Also signed homologue (SEQ ID NO:1), it can differently be expressed under the condition of Pi disappearance, and has separated from arabidopsis. The genomic clone of the psr1 of Arabidopsis is obtained (Figure 27 A-27B) also. For Southern blot analyzes, with DNA EcoRI (A), SacI (B), SalI (C), EcoRV (D) and BamHI (E) enzymolysis. The fragment screening-gene group library that trace is surveyed (Figure 27 A) and this 800 base-pair with 800 base-pair fragments of the 3 ' terminal that represents psrI cDNA has obtained genomic clone (Figure 27 B). The total band that all detects in both cases shows that this genomic clone represents the allele of psrI gene. Give the size with the band of probe hydridization.

Derivative arabidopsis gene coding is named as the polypeptide of psrPK (or psrI), it is the same with the polypeptide of black mustard, have and other the zone (seeing embodiment 9 and Fig. 8) of protein kinase height homology, and it is active to have serine/threonine (ser/thr) protein kinase. Yet arabidopsis psrPK is different from the protein kinase of former description with its black mustard homologue, because they have unique protein kinase C-terminal area. The zone of this uniqueness may relate to Pi concentration and detect, receives signal or transmission of signal, perhaps more than one of these functions. The substrate of protein kinase can be other composition of phosphate starvation response pathway, or the enzyme that relates in replying. These protein do not have obvious N-terminal signaling peptide, organelle target sequence or film interval region, and this just shows that they may bring into play function in the kytoplasm of cell.

The phosphorylation of protein kinase catalytic proteins substrate, and can in all biologies of living, find. Be known that they relate to adjustment process, wherein, phosphorylation/dephosphorylized function is the switch (or both are opposite) that activates/deactivate the substrate protein white matter. The protein kinase of some types relates to the replying of phosphate starvation of fungus and bacterium; Yet this is to show that the protein kinase of plant can be induced high-levelly for the first time under the Pi starvation conditions. Because psrPK is not having phosphatic period active especially, therefore, probably it plant to having the control action of switch in the replying of phosphate disappearance. PsrPK protein is expressed in the bacterium of carbon hunger with SNF1 homology, SNF1, and is shown as relating under this condition and controls metabolic response. Therefore, can change the expression in the related whole path of phosphate metabolism to the regulation and control of the kinase whose expression of psrPK, thereby produce valuable phenotype. The evaluation of the derivable gene of phosphate and characterization: β-glucosyl enzym psr gene

One group of homologue (psr3) of replying cDNA clone from the phosphate starvation of black mustard is confirmed as containing the β-glucosyl enzym (Malboobi and Lefebvre, 1995) take the part of peptide sequence as the basis. Encoding, this all provides among Figure 15 from the dna sequence dna (SEQ ID NO:7) of the β-glucosyl enzym (psr3.1B) of the next phosphate starvation induction of black mustard and the amino acid sequence (SEQ ID NO:8) of total length psr3.1B protein.

The engram analysis of the arabidopsis thaliana genomic dna of seeking and visiting with psr3.1cDNA shows that this gene only exists a position. Under the height stringent condition, corresponding genomic clone has been isolated in the screening in arabidopsis gene group library. Resulting clone is named as psr3.2 (SEQ ID NO:5), because its sequence generation is in the psr3.1 cDNA that is separated. The probe that the coding region of genomic clone is derived carries out engram analysis and shows that this gene has high-caliber expression in the root of Pi hunger, and is significantly improved in the root of having grown two days in the culture medium that lacks Pi. The expression of this gene is suppressed by thermal shock and anaerobic condition, and it can not obviously be induced by high salinity, or is induced by the disappearance of nitrogen and sulphur. The sequence analysis of this genomic clone disclosed to be had 13 extrons to exist and is interrupted by 12 introns that are rich in AT, also show the homology that has height with black mustard psr3.1B, and had high homology with the β-glucosyl enzym gene that comes from other kind. Similitude and otherness between the sequence of the amino acid sequence of psr3 clone's deduction and other β-glucosyl enzym advise that these genes will together be included in the genomic subgroup of BGA glucosidase of uniqueness with two other Brassicaceae gene. Have the endoplasmic reticulum stick signal in the carboxyl terminal and show that it is the cellularity position of psr3.2. Possible metabolism and the regulating and controlling effect of this enzyme in Pi hunger is replied is as previously discussed. The evaluation of the derivable gene of phosphate and characterization: other regulation and control psr gene

Dna sequence analysis and Computer Analysis to remaining nine psr clone are all carried out according to embodiment 8 described technology. The screening of the genomic library of other kind is by embodiment 9 described carrying out in order to determine these gene homologies.

The coding DNA of the glutamte dehydrogenase of the T3 part that resembles most in sequence from the dna sequence dna of the psr2 of black mustard, and at the ovomucoid (Figure 18) of the T7 of sequence part.

Resemble most the coding DNA (Figure 19) of coating protein from the dna sequence dna of the psr4 of black mustard.

Resemble most the coding DNA (Figure 20) of E.C. 2.7.2.4. from the dna sequence dna of the psr5 of black mustard.This sequence has shown the certain homology with the lysin coding DNA, and this lysin is another kind of E.C. 2.7.2.4., and it suppresses the signal conduction of cell cycle.

Dna sequence dna and coded protein from the psr6 of black mustard resemble the coding DNA of phosphoric acid salt transporton and coded protein (Figure 21) most.

The coding DNA of the histidine kinase of the T3 part that resembles most in sequence from the dna sequence dna of the psr7 of black mustard, and at the skeletal muscle calcium release channel albumen (Figure 22) of the T7 of sequence part.

Resemble most the coding DNA (Figure 23) of the translocator of sugar from the dna sequence dna of the psr8 of black mustard.

Resemble most the coding DNA (Figure 24) of adenylate cyclase from the dna sequence dna of the psr9 of black mustard.

The channel dna (G-protein) that resembles most calciphorin from the dna sequence dna of the psr10 of black mustard (Figure 25).

The coding DNA of the phosphatidyl inositol kinase of the T3 part that resembles most in sequence from the dna sequence dna of the psr11 of black mustard, and at three peptidyl peptase albumen (Figure 26) of the T7 of sequence part.

The separation of nucleic acid and structure

The invention provides the nucleic acid of separated DNA and reorganization, the protein of their encoded light synthesising biologicals or its functional protein, translating by the phosphoric acid salt shortage of DNA wherein or nucleic acid induced.In a preferred embodiment, protein wherein has the protein kinase activity, particularly serine/threonine protein matter activity, beta-glucosidase activity or phosphoric acid salt transporton activity.Term " nucleic acid " comprises DNA and RNA, also comprises strand and double-stranded kind.

DNA or RNA refer to from their source when being referred to herein as " separated " or origin (for example its existing cell, or in the nucleic acid mixture in its library) genomic dna or the cell RNA DNA or the RNA that separate, but also can be through further processing." separated " DNA or nucleic acid comprise the described method of utilization, similar method or other suitable DNA that method obtained and nucleic acid herein, also comprise purified basically DNA and nucleic acid, by DNA and the nucleic acid that chemical synthesis process produced, utilize biology and chemical process to combine DNA and the nucleic acid that is produced, and reorganization and separated nucleic acid.In this article, " reorganization " nucleic acid refers to the nucleic acid that the DNA method of utilizing reorganization is obtained, and comprises that those rely on the nucleic acid that method produced of artificial recombination, for example, and polymerase chain reaction (PCR) and/or to be cloned into carrier with restriction enzyme medium." reorganization " nucleic acid also refers to after process is introduced specified nucleic acid under the effect of cell nature recombination mechanism in cell, the result of the reorganization phenomenon that is taken place or produce.

Separated DNA comprises (a) SEQ ID NO:1, SEQ ID NO:3, or the nucleic acid that intercepts of the quilt of their coded proteinic functional parts of encoding; (b) has at least 80% homologous nucleotide sequence with SEQ ID NO:1 or SEQ ID NO:3, and the nucleotide sequence that is intercepted, it is encoded by the proteinic functional part coded with the 80% homologous nucleotide sequence of SEQ ID NO:1 and SEQ IDNO:3 at least; Or (c) nucleic acid of or hybridization complementary under medium stringent condition with any sequence (a) and (b).Term " functional part " refers to the proteinic part of psr, and it can influence the phosphate absorption and/or the metabolism of the nature (natural generation) of the proteinic photosynthetic organism of the inner endogenous psr of generation when expressing.Embodiment preferred intercepted dna sequence dna, it comprises the separated DNA of the 677-1020 Nucleotide that contains SEQ ID NO:1, the zone of the kinase whose uniqueness of coded protein.Also provide have 50% homology, preferred 80% homology, the more preferably DNA and the RNA of 90% homology, and under medium stringent condition with described DNA hybrid dna of claim 3 and RNA.The nucleotide sequence that also provides the quilt of above-mentioned DNA or nucleic acid to intercept, it has 10-20 or more a plurality of continuous nucleotides, and they can be used as probe or primer.

A cDNA of the present invention provides in Fig. 5, and it sign indicating number of reading that comprises 1020 Nucleotide is distinguished, and is combined with ATG initiator codon and TAG terminator codon, 339 amino acid of encoding.This cDNA further comprises 5 ' untranslated Nucleotide of 89 base pairs and 3 ' the untranslated Nucleotide of 141 base pairs, comprises 3 ' poly-A tail end to functional mRNA.The coded protein of this cDNA gives detailed introduction below.

Separated DNA further comprises: (a) SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11-17, SEQ ID NO:19-27, and the nucleotide sequence that intercepted by their quilt of any one coded proteinic functional part of coding; (b) have the nucleotide sequence that at least 80% homology is arranged with SEQ ID NO:5, SEQ ID NO:7, SEQID NO:9, SEQ ID NO:11-17, SEQ ID NO:19-27, and coding is by having the nucleotide sequence that the quilt of the coded proteinic functional part of the nucleotide sequence of at least 80% homology intercepts with they any one; Or (c) nucleic acid of or hybridization complementary under medium stringent condition with any sequence (a) and (b).The present invention also comprises by these nucleic acid encoded polypeptide.And then, also providing to protein with beta-glucosidase activity and with SEQ ID NO:5 has the aminoacid sequence of at least 80% homology or has the separated nucleic acid that the aminoacid sequence of 50% homology is encoded with SEQ ID NO:6, and to having the active protein of phosphoric acid salt transporton and the aminoacid sequence of at least 80% homology being arranged or have the nucleic acid that the aminoacid sequence of 50% homology is encoded with SEQ ID NO:18 with SEQ ID NO:17.10-20 with above-mentioned DNA or nucleic acid also is provided the nucleotide sequence that quilt individual or more a plurality of continuous nucleotides intercepts, and they can be used as probe or primer.

In this article, what term " homology " referred to is not to have identical origin, and is meant the similarity between the sequence.When determining the degree of the homology between two sequences, preferably relatively, as known in the art, compare the position between them with them.Occupied by same nucleotide amino acid when the position of being compared, then this two sequences is in this position homology.The homology of two sequences with the ratio that the homologous positional number is occupied in total position number of relatively crossing express, promptly express with percentage ratio.

Term " has the basic sequence homology " and should be understood that the sequence of being talked about has variation a little or non-significance, for example, and with SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:2, the slightly different sequence of SEQ ID NO:4.That is to say that the nucleotide sequence that " has the basic sequence homology " with SEQ ID NO:1 or the SEQ ID NO:3 essentially identical protein of in fact encoding for example, has the protein kinase of the regulatory function in phosphate metabolism.Predictably, some replacement and modification and don't the proteinic function of significance ground influence take place in the different piece at SEQ ID NO:1 or SEQ ID NO:3.The variation of sequence can be produced by sudden change.In addition, the sequence that has a homology with SEQ ID NO:2 or SEQ ID NO:4 will have the catalytic activity with the sequence similarity shown in SEQID NO:2 or the SEQ ID NO:4.In addition, also can have have protein kinase active SEQ ID NO:2 or SEQ ID NO:4 etc. the shape thing.For example, the sequence that has a basic homology with SEQ ID NO:2 can be the homologue that comes from other plant or species, for example, and from SEQ ID NO:4.Like this wait shape with homologous protein can be the immunology cross reaction.

Predictably, to having aminoacid sequence with SEQ ID NO:2 or SEQ ID NO:4 the protein of at least 80% homology or the nucleic acid of encoding with the protein that about 190-340 residue of SEQ ID NO:2 is the aminoacid sequence of about 50% homology are arranged, to produce the functional protein kinases, and the invention provides such nucleic acid.Have with the aminoacid sequence of SEQ ID NO:2 or SEQ ID NO:4 have an appointment 60%, 70%, 80%, 90% or the protein of higher homology can be also contemplated as and have the protein kinase activity.

The present invention also comprises because the degeneracy of genetic code and different with the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3 but coding has the active proteinic separated nucleic acid of protein kinase." degeneracy " refers to amino acid and has more than one Nucleotide ternary codon.For example, Methionin there are AAA and AAG.This is " tranquillization sudden change " (or the title " Wobble ") that occurs in the 3rd Nucleotide of codon, and coded amino acid remains unchanged.The present invention also comprises the sudden change that is not tranquillization and other modification, wherein, with 80% homology of SEQ ID NO:2 or SEQ ID NO:4, or all is retained with 50% homology of 190-340 the amino-acid residue of SEQ ID NO:2.

Like this, the present invention also comprise with SEQ ID NO:5-27 in any one has the aminoacid sequence and the nucleic acid of basic homology.

Those skilled in the art it should be understood that the present invention also comprises various forms of nucleic acid of the present invention, comprises the mRNA corresponding to cDNA of the present invention is sheared and the various forms that obtains.

The present invention further provides the height or the moderate stringent condition under be coded in the nucleic acid that at least a portion aminoacid sequence shown in SEQ ID NO:2 or the SEQ ID NO:4 or other psr protein or polypeptide are hybridized mutually.Stringent condition for hybridization is the term an of this area, refer to the actual temp that a nucleic acid and another nucleic acid hybridizes and the condition of buffer concentration, wherein, first nucleic acid can be and second complete complementary of nucleic acid, perhaps have between their two nucleic acid and be weaker than perfect complementarity to a certain degree [referring to Sections 2 and 6 in Current Protocols in MolecularBiology (Ausubel, F.M.et al., eds., Vol.1, Supplement 29,1995).The condition of hybridization is found in Maniatis et al., 1982 and Sambrook et al., 1989 description.

For example, the hybridization program of height stringent condition can (1) be used low ionic strength and pyritous washing, for example, 0.015M NaCL/0.0015M Trisodium Citrate, (0.1 * SSC) and 0.1% sodium lauryl sulphate (SDS) is arranged, temperature is 50 to pH7.0; (2) in crossover process, use Denhardt ' the s solution (high-purity bovine serum albumin of 0.1% weight/volume/0.1% weight/volume Ficoll/0.1% weight/volume polyvinylpyrrolidone) of 50% (volume/volume) methane amide and 5X, the 50mM sodium phosphate buffer, pH6.5, with 5 * SSC, temperature 42; Or (3) when hybridization, use 50% methane amide, 5 * SSC, 50mM sodium phosphate (pH6.8), 0.1% sodium pyrosulfate, 5 * Denhardt ' s solution, the salmon sperm dna of sonication (50 μ g/ml), 0.1%SDS and 10% T 500, temperature 42, washing are to be to carry out among 42 0.2 * SSC and the 0.1%SDS in temperature.

Change the condition of hybridization, the level that hybridization never takes place is changed to the level that hybridization at first takes place, can determine the condition that given sequence and the most familiar sequence are hybridized in sample.

The present invention also provides nucleic acid and the polypeptide of having been modified its structure by diverse ways, and these methods include but not limited to use the modification of transposon, and the site is the sudden change of property and randomness specially, and engineering Nucleotide replaces, disappearance or interpolation etc.

Protein

The invention still further relates to psr protein and psr polypeptide, for example, by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQID NO:11-17, the coded protein of SEQ ID NO:19-27, or by the coded protein of homologous nucleic acid.Shown in accompanying drawing, all psr polypeptide and homologue all are that the present invention is included.

Protein of the present invention and polypeptide can separated and/or reorganization.To refer to their separated and purifying be the state purer than native state in cell for " separated " protein and polypeptide herein.Embodiment preferred is not conform to other the protein or the protein and the polypeptide of the substantially pure of polypeptide." separated " protein or polypeptide comprise by herein method, similar approach and other suitable resulting protein of method and polypeptide, also comprise by resulting protein of chemosynthesis and polypeptide, combine resulting protein and polypeptide by biological method and chemical process, the protein of separated reorganization or polypeptide.Being called " reorganization " protein and polypeptide herein is by protein that expression of nucleic acids produced and polypeptide to reorganization.

Under state of nature, these proteinic translating are condition institute inductive that phosphoric acid salt lacks, and, protein has various activity, influence the level of endocellular phosphorus phosphate content and kind, for example, protein kinase, beta-glucosidase enzyme and phosphoric acid salt transporton activity.Preferably, these proteinic aminoacid sequences show has at least 80% sequence homology with SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:18, or has at least 50% homology with the amino-acid residue of the 190-340 of SEQ ID NO:2.

The separation of homologue

Nucleic acid provided by the invention or its part can be used for from contain coding to of the present invention on function the homologue of the photosynthetic organism cell isolating nucleic acid of the proteinic gene of psr of similar one or more.Term " psr protein " refer to its expression level under phosphoric acid salt shortage condition than under the competent condition of phosphoric acid salt, being induced into higher levels of protein.If cell has enough phosphoric acid salt, then this protein may not be expressed.In addition, this proteinoid is irrelevant with the mechanism that causes necrocytosis.

As described in the following embodiments, the inventor has utilized the black mustard suspension cell to separate nucleic acid of the present invention as parent material.In brief, the present invention has set up the cDNA library from lacking the parkerized black mustard suspension cell through 7 days.Utilization is not having phosphatic condition and is having two cover cDNA probes of the black mustard suspension cell generation of growing under the 5mM phosphoric acid salt condition that this library is screened.To those and the first cover probe strong hybridization takes place but do not separate with the clone that hybridization takes place the second cover probe.These clones' cDNA is inserted son carry out di-deoxynucleoside acid order-checking (Sanger, 1981) and determine its nucleotide sequence, and infer its aminoacid sequence thus.The psrPK gene is confirmed as the new protein kinase of can encoding.Other different hybridization, clone and sequencing technologies all is that those skilled in the art is known, can be used for obtaining the protein kinase gene that the inventor separates, other psr gene, and their homologue.

In addition, nucleic acid of the present invention can also be separated by following mode.The nucleic acid probe that utilizes the preparation of chemical synthesising technology or recombinant DNA technology to have at least a portion of SEQ ID NO:1 (or another psr sequence) or its homologue.This probe is carried out radio-labeling, screen cDNA library or genome dna library according to routine techniques with it.Can from black mustard, prepare, also can be from different or have the homologue that under suitable hybridization conditions, is identified or the genetically modified source and prepare.The DNA that identify in the screening library can use routine techniques cloning and order-checking.

The third method of separating nucleic acid of the present invention is to isolate or chemosynthesis goes out protein kinase polypeptide (or another psr gene), and utilizes this peptide to produce the anti-coded proteinic antibody of psr in animal.Then, with this antibody the cDNA library of setting up from black mustard or other source is screened, obtain the immune response sex clone according to routine techniques.With technology known in the art DNA from these clones is checked order then.

The 4th kind to nucleic acid of the present invention carry out isolating method be with DNA or RNA as template, utilize polymerase chain reaction (PCR) (Saiki et a1., 1985; Konat et al., 1991) optionally increase.When handling RNA, the method that this area that utilizes people such as Maniatis to introduce in nineteen eighty-two has been known can be separated total mRNA from cell.Be complementary cDNA with the enzymic synthesis of retroviral enzyme reverse transcription to mRNA then.Design and synthesize the Oligonucleolide primers suitable, in primer and cDNA mixture, carry out PCR with routine techniques (Innis et al., 1990) to being extended for of given nucleic acid.Can also comprise 5 ' and 3 ' RACE (rapid expansion cDNA terminal) method (Innis et al., 1990) in the PCR program.The dna fragmentation through expanding that is produced is cloned in the suitable carrier.

RNA molecule of the present invention can also be by will be suitable cDNA or made up in a kind of transcription vector that can obtain according to the above-mentioned cloned dna molecule that has increased.Generally, DNA is cloned into the downstream of promotor, and for example (downstream WI) adds suitable RNA polymerase (being the SP6 polysaccharase in this example) to the SP6 promotor of carrier pGEM 3Z, carries out responsive transcription according to the specification sheets of manufacturers for Promega, Madison.The promotor of general used transcription vector comprises bacterium phagocytosis T7 and T3 promotor.

The another kind of method of knowing of producing nucleic acid of the present invention or oligonucleotide is a chemical synthesis process.Well known various dna synthesizer, for example, by the Applied Biosystems in California Foster city, those instruments that the Millipore Corp. company in the Bedford city in Inc. company and Massachusetts week produces, they may be used to this class synthetic in.

Expression with the expression vector modifying protein of recombinating

Modification to psrPK or other psr protein expression can be passed through accomplished in many ways.In one embodiment, made up the nucleic acid of reorganization, but psrPK wherein or other psr protein be connected with the adjusting sequence by the place of working, for example, be connected with controlling gene expression level, time and tissue-specific promotor.

Can use conventional recombinant DNA technology to make up the expression vector of reorganization, this carrier can comprise the nucleic acid of the present invention of expressing protein kinase of the present invention or other proteinic at least a portion of psr.Referring to the description of people such as Sambrook in 1989.Constructed carrier can also comprise with the adjusting sequence of nucleic acid of the present invention " but the place of working is connected " expresses.In this article, term " adjusting sequence " comprise promotor, enhanser and known in the art other the control expression or the sequence of information stability.The known example that is suitable for the promotor of these purposes vide infra.What it will be appreciated by those skilled in the art that is, these examples are not determinate, and other promotor can be used to the phosphate absorption and the metabolism of photosynthetic organism.

In some cases, regulate composition tissue-specific expression can be provided.The adjusting sequence that refers to term " but place of working connection " contains expresses the needed competent composition of given nucleic acid, and this nucleic acid is correctly to be connected with the adjusting sequence.For example, nucleic acid of the present invention is in correct orientation, and with the initiator codon homophase.

In this article, term " promotor " and " promoter region " refer to dna sequence dna, be usually located at (5 ') upstream of the encoding sequence of structure gene, by to provide identification and binding site to control expression with the correct needed RNA polymerase of site transcriptional start and/or other the factor to coding region.

Promotor generally is divided into two classes, constructivity promotor and inducible promoter.Term " constructivity " represents not necessarily that in this article expression of gene all is identical level in all cells, and is meant that gene can be expressed in the cell of numerous types, though find that through regular meeting their richness is different.

Inducible promoter be can to inductor reply can be direct or indirect activation one or more dna sequence dna or the promotor of gene transcription.Do not have inductor, dna sequence dna or gene will not transcribed.Usually, combine the rho factor () of activated transcription all to be in the disactivation state with the evoked promoter specificity, then by inductor direct or indirect be converted into activated state.Inductor can be a chemicals, for example, protein, metabolite, growth regulator, herbicide or phenolic compound or because physical stress that hot, cold, salt, toxin etc. directly cause or the physical stress that causes indirectly owing to the effect of pathogenic agent such as virus and disease etc.Inductor can also be the illumination agent, the various character of for example illumination, dark, light such as wavelength, intensity, fluorescence, direction and time etc.The cell that contains inducible promoter can be exposed to these inductors by applied inductor by the outside, and for example, pair cell or plant are sprayed, water, heating etc.Come the expression of activated gene if desired at the specified time of plant-growth, just can apply inductor in that time.

The example of such inducible promoter comprises the thermal shocking promotor, for example the inducibility hsp70 thermal shocking promotor (Freeling, etal., 1985) of Drosphilia melanogaster; Cold inducible promoter is as the cold inducible promoter (White, et al., 1994) from colea (B.napus); And by alcohol induced alcoholdehydrogenase promotor (Nagao, et al., 1986).

In the known sequence that is used for providing structural genetic expression, regulation domain with Agarbacterium (Agrobacterium) gene-correlation is arranged, for example, nopaline synthase (Nos), mannopine synthase (Mas) and octopine synthase (Ocs), and the zone of regulating viral gene expression, for example 35S of Cauliflower embedded virus (CaMV) and 19S zone (Brisson, et al., 1984), be activated son (Takamatsu, et al., 1987) with the bag of TMV.

Other useful plant promoter is included in phloem and the dimension pipe portion camber expression promoter of plant, for example, glutamine synthase promotor (Edwards, et al., 1990), corn sucrose synthase 1 promotor (Yang et al., 1990), promotor (the Sagaya et al. that comes from the Rol-C of the TLDNA of Ri plasmid, 1989), and the phloem specific zone of pRVC-S-3A promotor (Aoyagi, et al., 1988).In addition, the plant promoter that can also use has little subunit (Coruzzi, et al., 1984 of Rubisco promotor (Rbcs); Broglie, et al., 1984) and the thermal shocking promotor, for example, soybean HPS17.5-E or HPS17.3-B (Gurley, et al., 1986).

Operable other promotor according to the present invention includes but not limited to following promotor:

(a) low temperature and ABA-responsive promoter, for example, Kin1, cor6.6 (Wanget al., 1995; Wang and Cutler, 1995), and from the ABA inducible promoter (Marcotte Jr.et al., 1989) of wheat EM gene;

(b) phloem specific sucrose synthase promotor, for example, from the ASUS1 promotor (Martin et al., 1993) of Arabidopsis;

(c) promotor of root and seedling, for example, ACS1 promotor (Rodrigues-Pousada et al., 1993);

(d) seed specific promoters, for example, 22kDa (Unger et al. from the zein of corn, 1993), ps1 Sugar receptors promotor (de Pater et al. from pea, 1993), from the phaseolin promotor (Frisch et al., 1995) of Phaseolus vulgaris;

(e) late embryo ample promotor, for example, lea promotor (T.L.Thomas, 1993);

(f) fruit-specific promoter, for example, from the E8 gene promoter (Cordes et al., 1989) of tomato;

(g) meristematic tissue specificity promoter, for example, PCNA promotor (Kosugiet al., 1995);

(h) pollen specific promoter, for example NTP303 promotor (Weterings etal., 1995);

(i) late embryo development-specific promotor, for example OSEM promotor (Hattori et al., 1995);

(j) for the ADP-glucose pyrophosphorylase tissue-specific promoter of guard cell and bulb parenchymatous cell, for example, from the ADP GP (Muller-Rober et al., 1994) of potato;

(k) conductive tissue specificity promoter, for example, from the Myb promotor of barley (Wissenbach et al., l993);

(l) the plastocyanin promotor in the light green tissue is for example from the plastocyanin promotor (Vorst et al., 1993) of Arabidopsis.

According to employed adjusting sequence, can be in whole or its part of plant by the nucleic acid of reorganization of the present invention institute plant transformed, and/or psr protein is carried out sufficient or inadequate expression at the different time of plant-growth.Therefore, can design starch and the accumulation of oil and other phenotypic characteristic etc. in the level of the relative size of the size of plant, each several part, flowering period, phytinic acid, the seed.

Known have numerous carriers that are suitable for the reorganization expressed in various cells.Can express psr protein to the carrier design of reorganization, for example, psrPK can carry out in prokaryotic cell prokaryocyte, for example, intestinal bacteria also can carry out in eukaryotic cell, for example, Saccharomyces cerevisiae (Saccharomyces cerevisiae) and Arabidopis thaliana, tobacco or canola etc.The expression vector of reorganization can be plasmid, bacteriophage or virus.The introducing of plant gene construction of the present invention can be used Ti-plasmids, the Ri plasmid, and plant viral vector, directly DNA transforms, microinjection, electroporation or the like is specifically referring to above-mentioned citing document.General expression vector all contains marker gene, in order to transformant is screened.There are some conventional carriers also to contain the sequence that at least a portion of another one functional protein is encoded, for example, Photinus pyralis LUC or bacteria beta-galactosidase.In the scheme of a this carrier of use, nucleic acid of the present invention is connected on this encoding sequence, produces fused protein, and it comprises the part of protein kinase of the present invention He another functional protein of at least a portion.Can be with designed carrier cell transformed according to the expression of luciferase, beta-galactosidase enzymes or other fused protein is screened.In addition, the protein of other that merges with psr protein can be invalid to screening, but can be effective to useful properties such as solvability etc. are provided, perhaps can be effective to protein purification scheme or industrial production etc., for example, the enzyme that in reaction, adds alcoholization.

Therefore, carrier of the present invention can be built as the form that contains the proteinic nucleic acid of coding psr, is used for various farm crop and gardening plant are comprised that unifacial leaf and dicotyledonous and gymnosperm etc. transform.The target of modifying can be whole strain plant, or certain tissue, the part of organ or plant, for example, seed.Furthermore, expression of gene be may be limited to a certain specific growth phase or envrionment conditions.The gene delivery system that is used to integrate these constructions will be according to the kind of selected target plant and difference; Yet, it will be appreciated by those skilled in the art that, existing molecular engineering can be used for successfully being modified with the farm crop of usefulness, for example, paddy rice, wheat, barley, oat, corn, soybean, Semen Brassicae campestris (canola), Sunflower Receptacle, oranges and tangerines, shaddock, lemon, potato, Radix Dauci Sativae, sweet potato, bean or pea, pea, romaine lettuce, tomato, Caulis et Folium Brassicae capitatae, Cauliflower, broccoli, radish, summer radish, spinach, onion, garlic, big capsicums, pumpkin, cucumber, apple, pears, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, rasp berry, pineapple, tobacco, banana, Chinese sorghum, sugarcane or the like.

For example, some plants such as rape (canola), tobacco and Arabidopsis have been carried out the genetic engineering processing, they have been contained can have the carrier that reduces or improve protein kinase quantity in whole strain plant or the seed that is produced.

In one embodiment, to the Arabidopsis cellular integration eight kinds of different constructions, they have structural (CaMV 35S) or seed-specific (Arabin) promotor.The description of these constructions sees the following form 1.

Table 1

Construction	Promotor	The arabidopsis thaliana protein kinases	Justice/antisense is arranged
				????1	Structural	Complete gene order	Justice is arranged
????2	Seed-specific	Complete gene order	Justice is arranged
				????3	Structural	Complete gene order	Antisense
????4	Seed-specific	Complete gene order	Antisense
				????5	Structural	The gene order of intercepting	Justice is arranged
????6	Seed-specific	The gene order of intercepting	Justice is arranged
				????7	Structural	The gene order of intercepting	Antisense
????8	Seed-specific	The gene order of intercepting	Antisense

Preceding four constructions that contain complete gene order provide in Figure 12 A-12D.

More particularly, Arabidopis thaliana is transformed by the construction described in the table 2.

Table 2:PSR1 construction and conversion process

	Construction	Be identified?	Transform (T1) [＞T2 seed]	Select T2 [＞T3 seed]
					????C1	CaMV+ has adopted psr1	Be	????A,B	????A:4,B:6
????C7	CaMV+ antisense psr1	Be	????A,B	????A:10,B:1
					????D1	Arabin+ has adopted psr1	Be	????A,B	????A:4,B:14
????D4	Arabin+ antisense psr1	Be	????A,B	????A:4,B:0
					????#2	CaMV+psr1 also has the sudden change ATP-binding site	Be	?????A
????#6	CaMV+psr1 also has the mutant kinase avtive spot	Be	?????A
					????#26	CaMV+psr1 also has the ATP site that has been removed Lys	Be	?????A
???psr1	CaMV+ has adopted psr1	Be	?????A
					???PA1	CaMV+ antisense 3 ' psr1
???PA3	CaMV+ antisense 3 ' psr1
					???AA5	Arabin+ antisense 3 ' psr1
??#2(P1+ ????P2)	CaMV+psr1 and at 250Thr site mutation (phosphorylation site)	Be	?????A
					??#12(P3 ????+P4)	CaMV+psrl and at 316 Ser site mutations (phosphorylation site)	Be	?????A

Used term and the total length of description C1 in pBI121 of construction had adopted psr1 and have the Cauliflower embedded virus in the table 2

The total length of 35S promoter (CaMV 35S) C7 in pBI121 has adopted psr1 and has CaMV 35S startup

The total length of sub-D1 in pBI121 has adopted psr1 and has seed-specific arabin

Promotor D4 in pBI121 total length antisense psr1 and have seed-specific arabin

No. 2 total length antisense psr1 that the sudden change ATP-binding site is arranged in pBI121 of promotor

And have the CaMV35S promotor (at the 33rd amino acids generation Lys

Change to Glu) in pBI121, the Lys disappearance is arranged in the ATP-binding site No. 26 (the 33rd

Amino acids) total length psr1 also has No. 6 total length psr1 that the mutant kinase avtive spot is arranged in pBI121 of CaMV35S promotor and band

Have the CaMV35S promotor (the 123rd amino acids take place Asp to

The change of Glu) psr1 in pBI121 total length psr1 and have the CaMV 35S promoter (with

C1 is equal to) No. the 2nd, 26,6, the 3 ' sequence and the psr1 of the uniqueness of 3 ' psr1175 base pair: on position transforms between promotor and gus gene: 170 strains/construction/transformation experiment, transformation experiment: A, B,

Transformant

The invention provides by the expression vector transformed host cells of reorganization of the present invention.In this article, term " by transforming ", " conversion product ", " conversion ", " transfected ", " transfection thing ", " transfection " all refer to and utilize a kind of in the known numerous methods of those skilled in the art that nucleic acid is incorporated in the cell.For example, the conversion to prokaryotic cell prokaryocyte generally is by making it " be easy to " accept foreign DNA with calcium chloride processing cell, then with such DNA and relevant cytomixis.Can also prokaryotic cell prokaryocyte be infected with the bacteriophage carrier of reorganization.

The method that introducing nucleic acid can adopt in more high biomass cells has virus infection, bacterium be media transfer (for example, Agrobacterium T-DNA transfer system), electroporation, coprecipitation of calcium phosphate, microinjection, lipofection, the nucleic acid bag is by the particulate blast technique, or other method, and specifically the kind according to cell determines.For draft section plant, for example, corn nuclear Chinese sorghum can be used the micro-injection blast technique, and for example, by Sanford, people such as J.C. are at United States Patent (USP) the 5th, 100, as described in No. 792 (1992).Other the operable method that vegetable cell is transformed sees that people such as Gelvin were description in 1992.Suitable pair cell transforms or transfection method can also be referring to the description of people such as Sambrook in 1989.Nucleic acid construct thing of the present invention can also be incorporated in the some concrete part of plant by method as herein described, sees for details following.

In order to help to identify the plant transformed cell, construction of the present invention has comprised further that also coding carries out the gene of plant selectable marker.The enzyme that provides antibiotic resistibility is provided useful selected marker, for example, gentamicin, Totomycin, kantlex etc. is had the enzyme of resistance.In the construction of Figure 12 A-12D and description in table 2, used NOS/NPT II kalamycin resistance gene to detect transfected vegetable cell.Similarly, but can also use the enzyme of the compound that the generation identification colors changes, for example, GUS (beta-glucosidase enzyme), or produce the luciferase of fluorescence.

The present invention also provides the DNA that will introduce to be incorporated into transformant in the genome, and the DNA that introduces is as the transformant of extrachromosomal element.Under latter event, can keep extrachromosomal composition with comparalive ease, promptly, introduce carrier then by bringing Selection In property mark in expression vector, under expressing conditions needed, marker gene allow cell grow.In one embodiment of the invention, screened as selected marker with the protein kinase activity by the expression vector cell transformed of reorganization of the present invention.Such cell can also be selected under the phosphate starvation condition.

In the experiment that in table 2, provides, provide Arabidopis thaliana conversion product as shown in figure 28 with the selection of kalamycin resistance process several generations.To the 2nd, 6 and No. 26 construction and psr1 construction, obtained T2 for seed from the conversion plant of beginning.C1, C7, D1 and D4 have been obtained the T3 of 10,11,18 and 4 kalamycin resistance plants respectively for seed.

The plant that contains nucleic acid construct thing as herein described and other photosynthetic organism have been the present invention further provides.Term " photosynthetic organism " comprises botanic member, comprises vascular plant and non-vascular plant (angiosperm, gymnosperm, fern, mosses, liver moss or the like), Cyanophyta plant (indigo plant-green alga, cyanobacteria), and photosynthetic bacterium etc.Suitable plant comprises monocotyledons and dicotyledons.Preferred monocotyledons is the crop with commercial value, for example, and paddy rice, corn, wheat, oat, sugarcane, Chinese sorghum etc.Preferred dicotyledons comprises Semen Brassicae campestris, Sunflower Receptacle, oranges and tangerines, tomato, broccoli, romaine lettuce etc.Also can use the algae conduct host cell of described construction herein.The example of algae has, Chlamydomonas reinhardtii, Chlamydomonas moewusii, Euglena gracilis, Porphyra purpurea, Cryptomonas sp. and Ochromonas sinensis.Prokaryotic organism also can be used as suitable host cells.Concrete example includes, Anacystis nidulans, Synechococcus sp., Rhodobacter sphaeroides, Rhodobactercapsulatus, Chloroflexus aurantiacus and Heliobacterium chlorum.

Construction of the present invention be directed in the plant, in the part of plant or in other the photosynthetic organism, operable technology has Ti-plasmids, the Ri plasmid, plant viral vector, directly DNA transforms, microinjection, methods such as electroporation.For these technology, can be referring to Weissbach and Weissbach, (1988), and Griersonand Corey, Plant Molecular Biology 2d Ed., Blackie, London, Ch.7-9 (1988).

The method that obtains transformant and/or aftergrowth is not a key of the present invention.Generally speaking, cultivated in the suitable culture base by the plant transformed cell, can contain the selection preparation in the substratum, for example, microbiotic uses selected marker to promote evaluation to the vegetable cell that has transformed.Selecteed transformant can be induced and be produced callus.In case formed callus, can promote the formation of twig with suitable plant hormone according to known method, then twig is transferred in the substratum of root and come aftergrowth.

The conversion product of the transgenic plant of Chan Shenging or its offspring can grow and the evaluation of productive rate like this.Can also estimate the content of phytinic acid, starch and the oil of the ratio of the features of blooming of transgenic plant, root and branch, seed.Can directly use these conversion products, also can further modify them with genetic engineering method or traditional method.

The transgenic plant that contain construction of the present invention can come from plant tissue with method well-known to those having ordinary skill in the art and method shown in Figure 28, the part of plant, or born again in the protoplastis.Therefore, the present invention includes cell, tissue (particularly meristematic tissue and/or embryonic tissue), protoplastis, epicotyl, hypocotyl, cotyledon, cotyledon knot, pollen, ovule, stem, root, leaf or the like.Plant can also regenerate from the explant of plant.Kind according to plant is used diverse ways.

Seed can obtain from the regenerated plant, also can produce with suitable intersecting of plant of the same race from the regenerated plant.In addition, the part of plant can be cultivated asexually propagated plant under the condition of suitable this partial regeneration.Then, can have the plant to the phenotype of phosphoric acid salt inductive psr protein active of having been modified, promptly can also can realize (seeing Figure 28) by seed growing by vegetative propagation technique with this plant repeat reproduction.

The homology dependent gene silence that comprises antisense nucleic acid

The present invention comprises that further homology relies on gene silencing, and it comprises the silence to the nucleic acid of the present invention of DNA-DNA pairing and RNA regulation and control.For the former, the interaction of nucleic acid that is introduced into or oligonucleotide and the homologue of self causes the common suspension of these genes.And the silence of RNA regulation and control comprises antisense technology, wherein, is in the nucleic acid of antisense or the cell that oligonucleotide is introduced into to nucleic acid of the present invention.Such antisense molecule can carry out base pairing (passing through hydrogen bonded) in antiparallel mode with nucleic acid of the present invention, the pair principle of the standard that is based on, that is, and G and C pairing, A and T or U pairing.Antisense molecule can comprise non-coding and regulating zone with the coding region or the non-coding region complementation of nucleic acid of the present invention, or with they all complementary (Gogarten et al., 1992; Shotkoski and Fallon, 1994).The codon of SEP ID NO:1 can be protracted or stride across in the complementary zone.Can comprise and regulate sequence complementary zone according to antisense molecule of the present invention, for example, but the non-coding and regulating regional complementarity that is connected with gene of the present invention place of working in expression constructs.In addition, can use the catalysis sense-rna (ribozyme) that the psr protein gene is transcribed to reduce expression of gene (Heinrichet al., 1993; De Feyter et al., 1996).Produce antisense molecule and can adopt chemosynthesis, PCR or expression vector etc., the technology of use can be described referring to prosthomere, or use conventional technology (Meyer, P.and Saedler, H., 1996).

The antisense construct thing includes but not limited to the following stated:

1. total length psr protein is being that opposite direction is placed with respect to promotor;

2. 3 ' zone (930-1272 Nucleotide) to the uniqueness of Fig. 7 is the complementary antisense sequences;

3. first three of the antisense sequences described in top 2/one (930-1080 Nucleotide) comprises the conservative nucleotide sequence of the part of coding EEEXXD sequence;

4. second 1/3rd (from the 1073 Nucleotide beginning) of the antisense sequences described in top 2, comprise the codon of conservative " D " residue of the 336th position of the aminoacid sequence of Fig. 8;

5. be the complementary antisense sequences to 3 ' the untranslated district up to polyadenylate polymerase (polyA) tail end (Nucleotide 1008-1240); Or

6. be the complementary antisense sequences to 5 ' the untranslated district (Nucleotide 10-88).

If under the condition that is suitable for seed-specific or structural promoter, have the death of the cell of antisense construct thing, then can use inducible promoter.There are justice and antisense nucleic acid to be transported in the cell according to of the present invention with one of any method well-known to those having ordinary skill in the art.

Isolated protein

The present invention also provides separated protein, they have protein kinase, beta-glucosidase enzyme, phosphoric acid salt transporton or other psr activity of proteins, particularly to the basic homologous aminoacid sequence of aminoacid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQID NO:10, the SEQ ID NO:18.In addition, separated protein of the present invention can be by coded with the nucleic acid of the nucleotide sequence hybridization shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11-17, the SEQ ID NO:19-27 under low or height stringent condition, or by the protein of any psr genes encoding.It should be understood that and the present invention includes the fragment that has homology and have psr protein active (being funtion part).

The proteinic molecular weight of the aminoacid sequence shown in the sophisticated not adorned SEQ of the having ID NO:2 is predicted to be 39,040kDa.Adopt the retrieval of pre-selected locations to determine the proteinic following various features of psrPK.It has serine/threonine protein matter kinase activity site between the 119-131 amino acid position, and between the 9-33 position ATP-binding site is arranged.Do not indicate it to have the long zone of any film relevant to the mapping of this protein hydrophobic, and the mapping of this proteantigen is shown that it has several zones that can be used as the polypeptide of producing this protein antibody with protein.They include but not limited to last 150 amino acid of C end.Owing to do not have tangible N end signal peptide and organoid target sequence, this protein to look like to be present in intracytoplasmic.The potential phosphorylation site that some amount is wherein arranged, they include but not limited to by the indicated position of the primitive of PKC, CK2 and as Fig. 9 shown in the Tyrosylprotein kinase phosphorylation site.They can the automatic phosphorylation site of yes or no.The glycosylation site that supposition is arranged at the C end regions.

Psr protein of the present invention can be by from cell or be secreted into wherein the substratum and separate.In addition, also can be as known in the art by chemosynthesis.As discussed above such, in this article, term " separation " refers to when producing with the dna technique of reorganization, does not contain other cellularity material or substratum, when producing with chemosynthesis, does not contain precursor.

The present invention also provides preparation to have protein or its segmental method of protein kinase activity, beta-glucosidase activity, phosphoric acid salt transporton activity or other psr protein active, this method comprises the steps: (ⅰ) but transforms with the expression vector pair cell of regulating the reorganization that the sequence place of working links together with containing nucleic acid of the present invention, (ⅱ) in the suitable culture base, cultivate by cell transformed up to forming psr protein, and (ⅲ) separate psr protein.Those skilled in the art can go out the scheme (Scopes, 1982) to isolated protein in other cell material and other the substratum according to the technical project of routine.These methods comprise the look popularize law, for example, and gel-filtration, ion-exchange chromatography and affinity chromatograph, and batch formula method of utilizing ion-exchange or affine resin.Utilizing ammonium sulfate precipitation, carry out resuspension and dialysis then, also is purification and the spissated scheme of using always.

In another embodiment of the invention, the fused protein that contains the functional part of proteinic at least a portion of the present invention can prepare with the step of aforesaid method.In certain embodiments, psr protein or its functional part can merge mutually with instructing fused protein excretory signal sequence from cell transformed.Utilize conventional technology will be come out by the excretory protein separation.

Antibody

The present invention comprises that also psr protein of the present invention is had specific antibody.The present invention includes mono-clonal and polyclonal antibody.In this article, term " antibody " comprises that psr protein is had specific antibody fragment, comprises that protein kinase, beta-glucosidase enzyme and phosphoric acid salt transporton etc. are had specific antibody fragment.Such fragment comprises that antagonist carries out the Fab fragment that proteolysis produces.In certain embodiments, antibody can be the antibody at the epi-position of the proteinic uniqueness of psr of the present invention.If protein is protein kinase of the present invention, so, these epi-positions can comprise the protein kinase avtive spot, ATP-binding site, and the C terminal sequence of 150 amino acid whose uniquenesses.

Complete psr protein or its immunity fragment can be used to prepare antibody.Antigenic protein is done in choosing or its fragment can be injected in the animal body (for example, rabbit, hamster, goat, mouse), causes that it is specific antibody that animal produces the antigen of being injected.Antigen generally all combines with adjuvant, for example Freund ' s adjuvant.In some cases, before injection, antigen elder generation and haptens or carrier molecule phase conjugate.Those skilled in the art can understand how concrete animal species is decided antigenic dosage according to its size, how in needs, to design reinjected scheme, how the antibody that produces is determined that it tires and how to separate in the serum of animal.The whole bag of tricks of preparation antibody that relates to the aspect of these aspects and other can see that Harlow and Lane were description in 1988.

When the manufacture order clonal antibody, at first gather in the crops the lymphocyte that produces at antigen, with conventional method (Harlow andLane, 1988) are merged in they and melanoma cells then.The hybridoma that do not go out that is produced like this screens the psr protein-specific antibody with conventional immune analysis method such as ELISA (enzyme linked immunological absorption detects).Separate these antibody according to method known in the art then.

Antibody of the present invention can physical property ground mutually coupled with any detection material well known in the art.These detection material comprise: radio isotope, fluorescein molecule, enzyme that can the catalysis colorimetric reaction etc.The example of such enzyme comprises alkaline phosphatase and horseradish peroxidase etc., and they all are that enzyme commonly used is detected in the laboratory.

Therefore, the invention provides in plant or plant part or other photosynthetic organism phosphoric acid salt is lacked institute's inductive protein, comprise the method that the expression of protein kinase, beta-glucosidase enzyme and phosphoric acid salt transporton detects, the step that this method comprises has (a) by causing the shortage of enough levels of available phosphorus to come the expression of induced protein to plant or plant part or other photosynthetic organism; (b) use to contact and the proteinic epi-position of antibody and this is combined and form antibody: antigenic mixture at this proteinic antibody and plant or plant part or other photosynthetic organism; And (c) detect antibody: antigenic mixture; Antagonist wherein: the detection of antigenic mixture is to be tell-tale to protein expression.

Transgenic plant and photosynthetic organism

The method of optimization plant characteristics is in the past all used traditional plant cultivation method, and wherein, the plant that has needed feature is hybridized produces the new real cultivation kind that has this feature.This method has two important defectives at least.At first, traditional plant cultivation method relies on the availability that has required feature in the known kind.And the needed feature relevant discussed above or be not the genomic part of live species and varient, or can not in the plant that has this feature really, can be expressed needed level with phosphoric acid salt.Secondly, traditional method of cultivation is a method relatively slowly.

The method of the feature of more modern modified plant (with other photosynthetic organism) is to utilize radiation or chemical treatment to come plant is produced sudden change.Such method is merely able to cause sudden change randomly in the DNA that forms Plant Genome, and sometimes this genome contains needed feature.Then the needed feature of foliage filter screening of sudden change is also cultivated subsequently.Though sudden change is better than natural selection because can mutate quickly in DNA of plants, it can not optionally take place preferable feature, and this method is at random.In addition, plant is exposed to the sudden change that mutagens can cause extra unwanted Plant Genome.Some such sudden change is not tangible immediately, therefore can't " be cultivated and get rid of " from the plant that contains needed sudden change yet.

Nucleic acid of the present invention and carrier can also be used to produce expression proteinic transgenic plant of the present invention and other photosynthetic organism.The genome of transgenic plant comprises the DNA transgenosis that has been integrated that is introduced among this biology or its ancestors.Contain the genetically modified DNA that is introduced into and to comprise the adjusting composition that is suitable for transfected biological or tissue.For example, when introducing as transgenosis, but nucleic acid of the present invention can regulate the sequence place of working with tissue specificity DNA and be connected, make protein kinase generation specifically in destination organization.Seed specific promoters will make protein expression only take place in seed.In addition, can also comprise 3 ' suitable zone, for example contain 3 ' zone of the polyadenylation signal of the Agrobacterium tumour of inducing (Ti) gene.Moreover, can also use 3 ' the suitable sequence of any characterization gene deutero-of plant or other biology such as animal, as long as they are considered to and can correctly bring into play its function in the environment of transgenic cell.In order to help the evaluation to transformed plant cells, construction of the present invention can further include the gene that needed plant selectable marker is encoded.

Therefore, genetic engineering provides to produce and has had the growth of having been modified, breeding, with the transgenic plant of metabolism composition and the method for other photosynthetic organism, in the cell of plant or other photosynthetic organism, introduce exogenous nucleic acid, its coding psr protein, as beta-glucosidase enzyme, protein kinase or phosphoric acid salt transporton, they take place to be transcribed under the condition of phosphoric acid salt disappearance in the natural species at its place, and their existence in transgenic plant or other photosynthetic organism can cause growth, breeding, modification with the metabolism composition, the cell or tissue that will contain exogenous nucleic acid remains under the condition of suitable this cell or tissue growth, can produce to have the growth of having been modified, breeding, with the transgenic plant of metabolism composition or other photosynthetic organism.Specifically, in the method, can use and contain the nucleic acid that comprises psrPK, psr3.2, psr3.1A, psr3.1B gene and homologue, be subordinated in the species of vascular plant such as angiosperm, gymnosperm, monocotyledons, dicotyledons, non-vascular plant and algae and produce transgenic plant.Therefore, the plant that has been enhanced of the activity that provides the phosphoric acid salt of natural generation to lack inductive protein kinase or beta-glucosidase enzyme and other photosynthetic organism.Because the gene that is introduced into is incorporated in the genome with being stabilized, so the present invention also provides and had by seed of the transgenic plant of decorative features and their offspring.

Use and use

In order to deal with the exogenous inorganic phosphorus of limiting growth quantity, photosynthetic organism some adaptability strategies that developed.But these strategies comprise the phosphatic availability of enhancing endogenous (Lefebvre et al., 1990; Sachay et al., 1991), more effectively use phosphorus to keep the most basic metabolism (Duffet al., 1994), and when enough phosphorus is arranged, excessive phosphorus be stored in vacuole (Lee et al., 1990; Mimura et al., 1990; Tuet al., 1990) so that when needing in the future, add to (Rebeille etal., 1983) in the tenuigenin.In addition, root system secretion acid and Phosphoric acid esterase improve phosphatic utilizability (Lefebvre et al., 1990 by discharge Pi respectively from rock phosphoric acid salt and phosphoric acid ester; Sachay et al., 1991).These alleviate the secretion (Goldstein, 1992) that mechanism relates to the change (Duff et al., 1991) of protein synthesis and degraded or the protein that has existed comprised Phosphoric acid esterase.They cause change (Rao et al., 1990 that rely on phosphatic reaction in light compositing; Usuda and Shimogawara, 1993), and breathing (Duff et al., 1989b; Duff et al., 1994; Hoefnagel et al., 1993; Nagano and Ashihara, 1993; Rychter and Mikulska, 1990; Theodorou and Plaxton, 1993), synthetic (Ashihara et al., 1988 of Nucleotide; Rychter et al., 1992), protein synthesis (Sadka et al., 1994), cell walls is synthetic and the change of other metabolite (Fife et al., 1990).

The physiology consequence of finiteness in black mustard (Brassica nigra) cell culture system to Pi carried out extensive studies.Comprise accumulation, starch and phenolic compound (Fife et al. to fat, 1990), raising (the Lefebvre et al. of the potential that Pi is absorbed, 1990) and to obvious distribution (Duff et al., the 1989b of the alternative enzyme that can " walk around " reaction that relies on Pi or Nucleotide; Duff et al., 1994; Theodorou andPlaxton, 1993).

Recently, for the internal stability environment of determining Pi concentration in the vegetable cell respectively regulate component, from Arabidopis thaliana, isolated excessive Pi be stored in mutant in the branch, i.e. pho2 (Delhaize and Randall, 1995).These authors advise that this sudden change can influence the adjusting of Pi being transported cytolemma.Another mutant pho1 of this kind of once identifying in the past is invalid (Poirier etal., 1991) for Pi being transported to xylem.

What advised is that nuclease and Phosphoric acid esterase successively cut and dephosphorylation (Goldstein, 1992) the RNA molecule in the Pi working cycle.What reported is that a kind of extracellular (Glund and Goldstein, 1993 are arranged in the tomato of Pi hunger; Being derived Nurnberger et al., 1990) (Loffler et al., 1992) with four kinds of intracellular nucleases.The active known of Phosphoric acid esterase is to be raised (Duff et al., 1989a in the plant that experience Pi lacks; Duff et al., 1991; Duff et al., 1994; Goldstein et al., 1989), and shown be acid phosphatase in the cell of the experience Pi hunger of the black mustard of cultivating, be synthesized (Duff et al., 1989b).Synthetic (Thedorou et al., 1992) of the α-subunit of the phosphofructokinase that PPi relies in the black mustard cell of experience Pi pressure, have also been induced.Recently, shown the phosphatase gene (vspB) (Sadkaet al., 1994) of having reduced sucrose induction at the Pi of soybean middle and high concentration.

Pi is hungry, and the inductive beta-glucosidase enzyme may relate to the de-glycosylation effect, regulates some enzyme (Ballou and Fisher, 1986 under Pi pressure; Gellatly et al., 1994).

Nucleic acid of the present invention, construction and method may be used to regulate photosynthetic organism to the replying of phosphate starvation, and these biological phosphate metabolisms.To the modification of psr protein active, promptly change or modify its expression level and activity, just can influence or change phosphate metabolism.Therefore, the efficient of phosphate metabolism can be enhanced, and feasible biology can not need to add phosphate fertilizer in lacking phosphatic soil and grow, even improves its growth in the competent environment of phosphoric acid salt.Reduce the required phosphate fertilizer amount of farm crop and/or improve its output and will produce significant and desired economy and environmental benefit.

Equally, can also modify some plant phenomenons of foundation phosphate metabolism.For example, those breedings be to depend in its environment in the photosynthetic organism of nitrogen to the ratio of phosphorus, the T/A of blooming can be modified.Bloom early will shorten the vegetative period of crop and reduce potted plant from seed to the time of blooming.On the contrary, for the plant of biomass as the results object, for example romaine lettuce and spinach, postponing that it blooms will be significant.

Modification to the phosphate metabolism of plant also can bring the change of the biomass ratio of its root and neck, and can be used for bringing economic benefit in the production of root crop such as Radix Dauci Sativae.In addition, will strengthen its drought-resistant ability in season at subsequently comparison exsiccant to plant in the ability of the bigger root system of the growth early evoking of plant.And then, if the growth of plant is the proteinic words of carrying out purifying subsequently in order to produce, so just can have tendency ground to express these protein in plant roots, the bigger root of plant of the present invention all is very useful for the biomass that improve root with the proteinic productive rate of being gathered in the crops.

Change cellularity Pi level and can also influence photosynthetic organism replying cold and/or frost.If the phosphoric acid salt of plant is restricted, then it has shown the growth in cold that has improved in the field.And then, having demonstrated inorganic pyrophosphatase and improved the sucrose level in the plant leaf, perhaps this point has partly reduced the level of Pi at the same time, thereby has improved the tolerance to cold and frost.Therefore, improving photosynthetic organism is to comprise suppressing or modification psr protein expression to one of method of the tolerance of cold and frost.

Another embodiment of the present invention is to improve Phosphoric acid esterase transporton protein expression level, for example expression of the psr6 in photosynthetic organism or its funtion part, thus improve the phosphatic absorption of environment.These protein can also be used among the application of plant adjusting.For example, the shortage of Pi can stimulate by regulating protein gene, thereby plant and other photosynthetic organism are absorbed owing to the deficiency to Pi and stores more Pi.

In addition, can also modify the vegetative organ that improves them and the nutritive value of organ of multiplication to photosynthetic organism.For example, spermatophyte such as Semen Brassicae campestris, soybean and corn etc. are that the form of phosphoric acid salt with phytinic acid (1,2,3,4,5, the salt of 6-hexamethylene six phosphoric acid) stored.When seed was raised as food with to animal, the existence of phytinic acid had just had problems.The animal of simple stomach can't metabolism phytinic acid and its phosphoric acid salt of use.In addition, phytinic acid combines with basic mineral substance, and for example calcium, magnesium, zinc etc. make them be difficult for comparatively speaking being utilized by animal.Can reduce the level of the phytinic acid in the seed to the modification of the phosphate metabolism of plant, phosphoric acid salt is stored with the form that is easier to be utilized by the animal and human.

The advantage of Compounds and methods for of the present invention is multiple, and those skilled in the art understands that the above-mentioned example that provides only is the part in the numerous application of the present invention.

The raising that protein kinase is expressed

The following examples will illustrate if psrPK is too expressed or modifies and improve its active words, what result to take place.A part of the present invention provides expression that opposite strategy makes gene or its homologue and is lowered, is modified or reduced their activity by " cancellation ".It will be appreciated by those skilled in the art that similar techniques may be used to modify other psr protein expression.

Generally speaking, can use the constructivity promotor to improve psrPK and the expression of similar gene in inverting biological, thereby improve the ability that they utilize phytinic acid, make their growths better in the environment that phosphoric acid salt is restricted, give plant or other biology can be in office grow in what phosphoric acid salt nutrient environment better capability.

Tissue specific expression in root can have the effect that increases the root size, improves root and obtain the effect of phosphatic ability from environment, and more effectively utilize phosphatic effect in the metabolism of root.

The specific expression of branch is expected to be and can changes the time and the degree of blooming, and can also improve in branch phosphatic utilising efficiency.

The expression of seed-specific can change phosphatic utilising efficiency in the seed, thereby forms more seed and make seed more full.Can also reduce simultaneously the content of seed mysoinositol six phosphoric acid.Can also improve starch and oily storage power in the seed.The black mustard suspension cell that phosphoric acid salt the lacks cell abundanter than phosphoric acid salt stores the carbon (Lefebvre et al., 1990) that more is fixed.

Other tissue-specific expression site comprises the coring that is used to improve phosphate absorption, and other since excessive or unsuitable expression can cause the pair cell adverse influence and cause the tissue of necrocytosis.One of purposes of this point is that generation is the male sterile line of purpose to be used to hybridize.

Reduce the method that protein kinase is expressed

The present invention includes the method for reduction to nucleic acid of the present invention and protein expression.For example, can produce genetically modified organism, its expressed molecule can be directly and endogenous psrPK protein bound, reduces its protein kinase active or itself and substrate or common factor bonded ability.And then, regulate the expression of molecule that composition or trans-acting regulatory factor combined and disturbed their function with cis acting, can reduce the expression of protein kinase.

The third method can be used nucleic acid or proteinic modification, makes them be gone functionalization or disturbed to original (natural) functional protein can reduce or cancel the active mode of its protein kinase.

The 4th kind of method can be used the described antisense molecule of superincumbent second joint.The inventor thinks, when antisense molecule of the present invention is sent in the target plant cell, it will combine by the kinase whose endogenous nucleic acid molecule of hydrogen bond and coded protein, thereby reduce the genetic expression of this protein kinase.Antisense molecule can be designed as the initiator codon that itself and endogenous protein kinases coding nucleic acid molecule complementary zone comprise sense strand.Antisense molecule can comprise that both are the complementary zone to the coding region of sense strand or non-coding region one or they.

The 5th kind of method can be used conversion, the nucleic acid of being introduced comprises with the whole of the nucleic acid of the present invention of sense orientation arrangement or its a part of, reduces the expression of protein kinase, can be any mode, comprise that gene suppresses (Meyer and Saedler, 1996) altogether.

Though the present invention has been referenced the mode of optimum implementation and has been described in detail,, these embodiments are all just illustrative, are not restrictive.Adopt principle of the present invention, and the spirit and scope of claims of the application who does not break away from, can also design other embodiment.

All documents of being quoted from are all by being incorporated in this paper in this citation.

Embodiment

Embodiment 1: the cultivation of vegetable cell

In the MS substratum according to described (Lefebvre et al., 1990) cultivate non-photosynthetic mushroom black mustard suspension cell, this MS substratum (Murashige andSkoog, 1962) contain 6% sucrose (17.5mM) and 2mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), with 24 degrees centigrade and per minute 130 commentaries on classics shakes cultivations.In order to guarantee that all are in same nutritional status by cultured cells, the culture of 6 milliliters 7 ages in days that contain 3 ml cells (cell volume of consolidation) is inoculated in 44 milliliters of fresh MS substratum that contain 1.25mM Pi.The determining cell rested in the aseptic gradient tube after 45 minutes of the cell volume of consolidation measured.Cultivate after 7 days, the cell of same quantity is joined 44 milliliters of fresh containing in 10mM, 1.25mM phosphoric acid salt or the not phosphatic MS substratum cultivated again 7 days.For the processing of 10mM Pi, to the KH of sterile filtration ₂PO ₄PH is adjusted to 5.8, KOH is joined in the substratum of 1.25mM Pi.In the processing that does not have Pi, weight-molal KCl such as use to substitute KH ₂PO ₄

Fig. 1 has provided growth and the feature endogenous phosphate concn.The cell that has added 10mM Pi all has competent nutrient between whole incubation period.Added the cell of 1.25mMPi, compared with the cell that does not have Pi to handle, only experienced gentle Pi disappearance, it relied on is phosphatic conversion to inside.Quantitative assay to endogenous acid phosphate was carried out (Lefebvre et al., 1990) according to former description.

Grow after 7 days, fritted glass filter is collected cultured cells, and the aperture is 10 microns, with 100 milliliters 0.5mM CaCl ₂Washing is stored in-70 degrees centigrade the liquid nitrogen then until use.

Embodiment 2: extract total RNA and mRNA

From the black mustard cell of the embodiment 1 of results, isolate total RNA according to the described method of Chirgwin (Chirgwin et al., 1979).Carry out homogenate at ice-cold temperature pair cell, the homogenate of use contain 4M isothiocyano guanidine (GIBCO/BRL, Burlington, Canada).(NJ) program of being recommended is to poly (A) for Uppsala, Sweden and Piscataway according to Pharmacia company to use the mRNA purification kit ⁺RNA (mRNA) purifies.To the total RNA and the total mRNA that are extracted in never Pi processing, the cell that handle with 1.25mM Pi and that 10mM Pi handles, the optical density meter is quantitative, and checks (Sambrook et al., 1989) with formaldehyde/sepharose.

Embodiment 3: with the analysis of external translation to the change of the colony of mRNA kind

What known is that the situation with the protein synthesis black mustard cell that gives Pi Pi hunger is different.This difference of replying to Pi hunger can be regulated and control by several different methods.For whether the change of determining protein level is caused by the variation of the level of translating, to hungry from Pi and added the external translation of mRNA that the black mustard cell of Pi extracted and compare.The external translation of the mRNA of above-mentioned purifying is according to Promega (Madison, WI) program is carried out, used wheat extract (Promega) and (35S)-methionine(Met) (ICN, Costa Mesa, CA) and be diluted to ultimate density O.5mCi/ml with unlabelled methionine(Met).

After the translation, from each reaction mixture, take out the sample aliquot of 5 microlitres, with the gel loading damping fluid merging of 20 microlitres, the little 1%SDS of ultimate density (Laemmli, 1970).Sample is placed in the boiling water bath 5 minutes, carry out sex change SDS-polyacrylamide gel electrophoresis (SDS-PAGE) then, that use is LKB 2010 Macrophor electrophoresis apparatus (Pharmacia Biotech, Inc., Baie d ' Urf é, Canada) and the discontinuous system of Laemmli (Laemmli, 1970).In the electrophoresis standard of parallel molecular weight to alpha-lactalbumin, carbonic anhydrase, Glyceraldehyde-3-phosphate desaturase, egg white powder and the bovine serum albumin of 14C-mark be respectively 14,29,36,45 and 66kDa (Sigma, St.Louis, MO).Gel is the thick blob of viscose of 0.4mm that contains 1%SDS.The concentration of acrylamide monomer in stacking gel is 5% (w/v), and the concentration in separating gel is 10% (w/v).The length of separating gel is 35cm, isolated protein to greatest extent.Electrophoresis is to be that 30mA carried out 5 hours at 25 degrees centigrade with the constant current.

After electrophoresis is finished, be combined in the gel of using in conjunction with on (Pharmacia) sheet glass of brine treatment, in containing 600 milliliters of stationary liquids of 20% trichoroacetic acid(TCA) (TCA) and 10% methyl alcohol, cultivated 20 minutes.Clean gels three times with containing 10% ethanol and 5% acetate 600 milliliters fixing back washing lotions then, in stink cupboard, allow this sheet glass dried overnight under the room temperature.The dry gel of crossing is strengthened instrument at-70 degrees centigrade with Cronex, and (DuPont, Wilmington DE) to the exograph exposure, use Kodak film processor and solution to wash out egative film.Use the visual inspection radioactive automatic developing, and scan with LKB enhanced UltraScan XL laser intensity meter (Pharmacia).Result to densometer scanning analyzes with GelScan XL software second edition (Pharmacia).

The result who analyzes provides in Fig. 2.For easy, the density meter of each band is shown percentage ratio at external synthetic polypeptide, therefore, also be percentage ratio to the mRNA of coded polypeptide.The difference of the density of each band that various Pi handle is considered to represent the difference of the richness of the sort of mRNA that produces this band.But, should be borne in mind that since protein by ³⁵S-methionine(Met) mark, so signal is the percentage ratio to the methionine(Met) of every peptide species, in order to compensate this factor, only the signal with same molecular weight compares between different processing.

Most ³⁵The translation product of S-methionine(Met) mark all has been detected among various Pi handle.Among three kinds of processing, have 16 peptide species, strength of signal is had nothing in common with each other.The data of the polypeptide expression difference that Fig. 3 has summed up never, and Pi handles, produce in the cell that handle with 1.25mM Pi and 10mM Pi processing after by stdn.On the basis of these analyses, the shortage of Pi has caused mRNA template number to be enhanced in ten peptide species, is lowered in other six kinds.In showing the interpretable mRNA of adorned expression, four kinds corresponding to 31.7,32.3,52.5 and the protein of 64.8kDa molecular weight all only in the processing that Pi lacks, be detected.Compare with other polypeptide, they Pi lack during in by with higher relatively horizontal expression.In the processing that lacks Pi, be also noted that inhibition to the polypeptide of 43.5kDa.The molecular weight of the polypeptide of the mRNA that other six kinds of representatives are preferentially expressed between the Pi hunger period estimates to be respectively 19.6,40.6,30.1,37.0,18.6 and 29.5kDa.Their expression in the cell of handling with 10mM Pi of the expression ratio in the processing that Pi lacks have improved 3.3,2.8,2.7,1.7,1.3 and 1.2 times respectively.In addition, to molecular weight be estimated as respectively 14.4,35.9,48.3,18.4 and the expression in the processing that Pi lacks of the polypeptide of 35.5kDa reduced by 20,6.6,1.8,1.6 and 1.4 times respectively.In the cell that 1.25mM Pi handles, to the mRNAs of 16 kinds of polypeptide expressed codings well as if with medium level exist or with to lack level similar in handling at Pi and exist.

For using total RNA or polyadenylic acid RNA, do not seek and visit difference between the result to the gel electrophoresis of external translation product.Above-mentioned result is consistent in three are independently tested.

Embodiment 4: construction cDNA library from the black mustard cell that lacks the Pi processing

With the about 10 microgram polyadenylic acid RNA that under lacking the Pi condition, cultivated 7 days cell, purified, according to the specification sheets of manufacturer with c-CLONE II cDNA synthetic agent box (Clontech, Palo Alto, CA) synthetic double chain cDNA library.(Bio/Can Scientific, Mississauga Canada) separately, obtain the cDNA molecule of length greater than 400 base pairs to the pillar of the cDNA product process Chroma Spin-400 of the widow-d that is produced (T)-ending.The length of these molecules is estimated between about 0.5kb-3kb.Use the viscosity tail end of EcoRI restriction enzyme site, recommend according to manufacturer that (Stratagene, La Jolla CA) will be connected to by EcoRI in advance in the arm of the enzyme λ ZAP II bacteriophage cutting/handled by Phosphoric acid esterase according to the cDNA molecule that length is selected.Use Stratagene ' s Gigapack II Gold to pack the phage of reorganization, be titrated on the lawn of the intestinal bacteria XLl-cyanobacteria that grows on the LB agar disks according to the program of the recommendation of manufacturer.At best cDNA: (44ng: joint efficiency 500ng) is 7.5 * 10 to the carrier ratio ⁶Plaque-forming unit (pfu)/μ g cDNA.Embodiment 5: differential is carried out in the cDNA library that Pi is lacked the black mustard cell of handling

Hybridization and cross hybridization screening

Differential screening is carried out with the hybridization from the cDNA probe that comes with the black mustard cell that has added 10mM Pi that lacks that Pi handles in the cDNA library of the black mustard cell of handling from above-mentioned shortage Pi, to identify the gene that the difference of the protein of these cells and mRNA is responsible for.

The mRNA that the cDNA probe of strand uses the cell handled from Pi disappearance and 10mM Pi is synthetic, prepares respectively-P and+P probe.Concrete way is (Sambrook et al., 1989) as mentioned previously, used the mRNAs of 5 micrograms and six deoxynucleotides at random (Pharmacia) of 7.4 micrograms.Radiolabeled adding be by in reaction mixture, add α- ³²P-dATP (ICN) and realize.Use phenol: the chloroform extraction reaction product, purify with sephacryl gel S-300 post (Pharmacia).

The cDNA library of not amplification is added on the lawn of the blue fungus strain of intestinal bacteria XLl-of growing with low density (2,000pfu/90mm LB culture dish).Screen as follows to ading up to 50,000 plaque: at first, the extractives of heavy part of preparation from each culture dish.With the DNA sex change of plaque and with ordinary method (Sambrook et al., 1989) be immobilized in the Nitrocellulose strainer (Amersham, Oakville, ON, Canada) on.This strainer is cured, prewashing is 1 hour in the solution of 5X SSC, 0.5%SDS, 1mM EDTA (pH8.0), the solution of salmon sperm dna (Pharmacia) 6X SSC, 0.05X bovine lacto transfer technique optimizer, 25 μ g/ml sex change, fermentation in 68 degrees centigrade of following prehybridization 2-3 hours [the 1X bovine lacto transfer technique optimizer contain 5% fat free dried milk (Carnation Inc., Toronto) and 0.02% sodiumazide (Sigma)].In the strainer of each counterweight part, one with radioactivity-P probe and another and radioactivity+P probe 68 degrees centigrade of hybridization of spending the night.Wash these strainers then, condition is that to improve tight degree step by step as follows: give a baby a bath on the third day after its birth time, and at 2X SSC, under the room temperature among the 0.1%SDS 5 minutes; Wash twice, at 1X SSC, among 68 degrees centigrade 0.1% the SDS 1-1.5 hour; Wash once, at 0.2X SSC, 1 hour (containing 0.15M NaCl and 15mM trisodium citrate in the 1XSSC solution) among 68 degrees centigrade 0.1% the SDS.Radioactive automatic developing carries out according to top description.With-P probe hybridization but the clone with+P probe hybridization is not confirmed as " positive ".Then, these clones are carried out second screening of taking turns with third round.

Through the screening of three-wheel, 131 preferential clones that express in hungry cell have been obtained.Because strengthened the overall number (so that not missing any one) of screened plaque wittingly, these 131 clones have comprised the multiple clone of same gene.Therefore, be necessary the clone who is separated is carried out cross hybridization, distinguish new clone and multiple clone.

Separated clone carries out enzymolysis with EcoRI, PstI and TaqI restriction enzyme, and resulting dna fragmentation separates on 1% sepharose, transfers to Nytran film (Schleicher﹠amp; Schuell, Keene NH) goes up spend the night (Sambrook et al., 1989) in the SSC of 10X. ³²The single-stranded probe of P-mark inserts system fully from single clone's the black mustard that is released, and adopts PCR (Konat et al., 1991) method, uses T in first round reaction ₃And T ₇Primer is taken turns second and to be used T in the reaction ₃Or T ₇Primer.

The DNA institute bonded film of separating from the clone cured under 80 degrees centigrade 30 minutes earlier, then at 65 degrees centigrade 0.25M NaH ₂PO ₄(pH7.2), prehybridization 5 minutes among 7%SDS, the 1mM EDTA.Then, add radiolabeled probe, hybridized 2 hours.With film at 40mM NaH ₂PO ₄(pH7.2), washed twice among 5%SDS, the 1mM EDTA, again with film at 40mM NaH ₂PO ₄(pH7.2), washed twice among 1%SDS, the 1mM EDTA, each 30-60 minute, temperature was 65 degrees centigrade.Radioactive automatic developing carries out as mentioned above.In the SDS of 0.1X SSC and 0.5%, the bonded probe is discharged from film in 95 degrees centigrade of 20 minutes washed twice.

The result that cross hybridization is analyzed can be in 11 different homology groups Pi inductive clone's great majority, and their are named to " Pi hunger is replied " (psr) organizes 1-11.Because these genes phosphoric acid salt lack during in active especially, can think that they relate to plant among the replying of phosphate starvation.Embodiment 6: use the RNA blotting that psr cDNA clone is carried out RNA and analyze

Inductive psr mRANs and richness thereof are further analyzed with the RNA blotting under different phosphatic growth conditionss, and its result provides in Fig. 4.

Clone's psr cDNA inserts body and function EcoRI enzymolysis and discharges from carrier DNA.Reaction product is walked electrophoresis in 1% sepharose, the band that will contain the DNA of insertion shears off.Subsequently, (Bio 101 Inc., Vista CA) purify, with random primer reaction carrying out radio-labeling (Sambrook et al., 1989) with the GeneClean II to this DNA.(Clontech, Palo Alto CA) purifies to radioactive probe with Chroma Spin-30 post.Contrast dna probe (alpha-tubulin) carries out similar radio-labeling and purification.

That 30 micrograms lack to be handled from Pi, added 1.25mM Pi and added the total RNA that extracts the cell of 10mM Pi and carried out electrophoresis (Sambrook et al. at 2.2M formaldehyde/1% sepharose, 1989), the program jump according to manufacturer arrives NytranPlus film (Schleicher﹠amp; Schuell) on.

Containing 50% methane amide, 0.12M NaH ₂PO ₄(pH6.8), cultivated one trace 30 minutes in 42 degrees centigrade in the prehybridization damping fluid of 0.25MNaCl, 7%SDS and 1mM EDTA.They are transferred to contain 2-5 * 10 ⁷In the fresh hybridization buffer of cpm probe, the activity specific of probe is about 4 * 10 ⁹Cpm/ μ g.Hybridization is spent the night at 42 degrees centigrade and is carried out, and then in 0.1% SDS of 2X SSC, room temperature, among 0.1% the SDS of 0.5X SSC, room temperature, and washs respectively 30 minutes in 0.1X SSC, 65 degrees centigrade 0.1% SDS.Radioactive automatic developing carried out 1-4 days.In order to reuse trace, the bonded probe was washed in 0.1% SDS 5 minutes, and balance is 20 minutes in the 5X of room temperature SSC, 0.1% SDS, peels off 2 minutes in 95 degrees centigrade same solution at last.

According to comparing with the internal standard of alpha-tubulin, most psr gene is all expressed to relative height, and lack to handle at Pi with the cell that has added 1.25mM Pi in all induced, just the expression in the latter is a bit weaker.In the cell that has added 10mM Pi, observed psr7,8,9,10 and 11 low-level expression, and do not detected gene transcription other.Highly tight washing will not remove with extra the taking that psr9,10 and 11 all Pi as probe see in handling.For them, the mRNA that differential is expressed is minimum in the expression that is detected.

Embodiment 7: the experiment out of control of nuclear

Make the plasmid DNA sex change of the alpha-tubulin insertion body that contains black mustard psr1 cDNA or contain Arabidopis thaliana with alkali, use Bio-Dot micro-filtration equipment (Bio-Rad) in that 5 micrograms/naming a person for a particular job, it is added to (Schleicher and Schuell on the Nytran-Plus film then, Guelph, Canada).In order from new synthetic mRAN, to produce probe, from the above-mentioned interpolation of 5-6 gram isolate the nuclear (Willimizer and Wagner, 1981) of transcriptional activity 5mM Pi and root tissue Pi hunger.Adopt the method for Chappel andHahlbrock (1984), have at 30 degrees centigrade ³²Transcribed 60 minutes under the condition that P-UTP exists.RNA to mark purifies according to the described method of people such as Somssich (1989).In hybridization, used about 10 according to described (Malboobi and Lefebyre, 1995) method and dot blotting ⁶The rna probe of cpm.After the washing, the rnase in 2X SSC with 20 μ g/ml under the room temperature was handled 30 minutes at last.Then trace is washed twice with 2X SSC, 0.5% SDS, wash twice with 2X SSC.Dot blotting is to exograph exposure 7 days or longer.The result provides in Figure 10.

Embodiment 8: plant culture

The seed of Arabidopis thaliana (Columbia strain) carries out surface sterilization in 30% SYNTHETIC OPTICAL WHITNER (Javex), 0.03% Triton X-100 (Sigma), then with sterilization washing six times.Seed transferred to be placed on contain 0.5% agar and examine every lattice 1cm on the solid MS of 2% sucrose (the Murashige and Skooge) dish ²The iron grid in.After 11 days, seedling is longer, and root passes the iron net, they are transferred in the liquid MS medium of half intensity of 15ml, and this substratum contains 1.25mM Pi and 1% sucrose, is arranged in the erlenmeyer flask of 125ml, temperature is 24 degrees centigrade, and illumination is 540 Luxs, changes the medicines cultivation of shaking with per minute 80.After three days, plant is transferred in the MS substratum that contains various following concentration nutrients.For the processing of the Pi of various different concns, with the KH of filter-sterilized ₂PO ₄With KOH pH is adjusted into 5.8, joins in the substratum of 1.25mM Pi, reach 5mM.For the treatment group that lacks Pi, with the alternative KH of the KCl of filter-sterilized ₂PO ₄, reach 5mM.

Embodiment 9:DNA order-checking and Computer Analysis

According to supplier's guidance (Stratagene), external from the λ ZAP II phage of the reorganization selected, shear under psr cDNA, under the condition that has R408 to help phage to exist, put in the BluesciptTM plasmid vector.Plasmid DNA prepares (Sambrook et al., 1989) with the CsCl gradient centrifugation.

(T is used in OH) order-checking to each bar chain of separated DNA for United States Biochemical Corp., Cleveland with Sequenase Version 2 Kit ₃And T ₇Primer, and other to primer (Stratagene) that should sequence.5 ' and the 3 ' order-checking of holding to these clones has shown the difference between the psr protein in 11 immediately significantly.

Research to the homology of the sequence that obtained is carried out the GenBank database by NCBI (national bioinformation center) with BLASTX program (Gish etal., 1993).Assembling and editor's encoding sequence, and use to the LASERGENE related software of Macintosh come analyzing DNA and prediction protein sequence (DNASTAR, Madison, WI).

Clone psr1 be confirmed as the encoding gene of phosphate starvation inductive protein kinase.Therefore, give the name of its another one, psrPK.This psrPK sequence has the high homology with the protein kinase of separating from other plant, for example, Glycine wax protein kinase 2 (SPK2, GB registration number L19360,63% homology), arabidopsis thaliana protein kinases 2 (ASK2, GB registration number Z12120,70% homology) (Park et al., 1993), colea serine threonine kinases 1 (BSK1, GB registration number L12393,69% homology), Glycine wax protein kinase 3 (SPK3, GB registration number Ll9361,76% homology), colea serine threonine kinases Z (BSKZ, GB registration number L12394,71% homology), arabidopsis thaliana protein kinases 1 (ASK1, GB registration number P43291,71% homology) (Parket al., 1993).

From obtained the λ PRL2 cDNA library (CD4-7) of Arabidopis thaliana from Arabidopsis Biological resources center, Ohio.Under the height stringent condition, it is screened with the probe that contains black mustard psrPKcDNA insertion body.The body and function of inserting black mustard psrPK cDNA causes the synthetic of probe at random, and guidance subsequently according to manufacturers (Du PontNEN, Boston, MA) at on-radiation Du Pont Renaissance ^TMHybridization in the test kit.The plaque (called after psr1-1) of strong hybridization is purified out, and DNA is separated for its bacteriophage.After shearing from the Bluescript plasmid in vivo, the Arabidopis thaliana of this phage inserts body to carry out the di-deoxynucleoside acid of every chain is checked order.

Fig. 5 has provided the cDNA sequence of black mustard psrPK, arranges with the kinase whose cDNA sequence alignment of the corresponding proteins matter of coming from Arabidopis thaliana.

Embodiment 10: the separation of genomic clone

Probe with the psrPK cDNA insertion body that contains Arabidopis thaliana under the height stringent condition screens the arabidopsis gene group library in EMBL3.This insertion body and function causes the synthetic of probe at random, and the guidance according to manufacturers subsequently (DuPontNEN, the hybridization in on-radiation Du Pont RenaissanceTM test kit Boston).Positive colony is continued screening until homology, carry out the southern blotting technique analysis with above-mentioned probe then.Insertion body from positive colony is sheared and digestion with suitable Restriction Enzyme, and subclone is in plasmid then.To insert body and function di-deoxynucleoside acid sequencing then checks order to two chains.

Embodiment 11: carry out Plant Transformation with the psrPK construction

Arabidopis thaliana is transformed with eight kinds of different constructions in plant, used constructivity and tissue-specific promoter, be connected on the psrPK sequence from Arabidopis thaliana whole or part of justice or antisense.See Figure 12 A-12D, table 2 and Figure 28.

At some construction (C1, C7, D1, D4, PAl, PA3 and AA5, see Table 2) in, be present in the gus gene (CLONTECH in the pBI121 carrier, PaloAlto, CA, www.clontech.com) had (psr1) (Figure 12 A) of justice or antisense (α-psr1) (Figure 12 B) psrPK gene replaces, and is created in the construction that justice or antisense arranged of structural (CaMV-35S) promotor under controlling.The 35S promoter of pBI121 (Cauliflower embedded virus 35S promoter) is fused to the upstream of gus gene.Cut with SamI and EcoICRI enzyme gus gene is removed from carrier.The cDNA of coding psrPK is cloned in the pZL1 carrier and (is separated from the cDNA library at Arabidopsis resource information center).With SmaI and BamHI enzyme this clone being carried out enzyme cuts.Insert the Klenow fragment of dna polymerase i subsequently in the BamHI site.Resulting carrier carries out blunt end with the psrPK gene and is connected.The result is, obtained being subjected to the control of CaMV 35S promoter the psrPK gene justice and antisense construct thing arranged.In other construction [the 2nd, 6 and No. 26 psr, No. 2 (P1+P2), No. 12 (P3+P4)], psrPK directly is cloned into the BamHI/SmaI site of pBI121, between promotor and gus gene, need not remove the latter.

With connecting product suitable bacillus coli DH 5-α is transformed, and be placed on the LB/kan plate culture medium.Choose the bacterium colony and the microtitre of growth.Distinguish the construction that justice and antisense are arranged with the enzymolysis that SalI and EcoRI enzyme carry out, be respectively 1.6kb and 0.3kb.

The construction that provides in Figure 12 C and 12D prepares with similar methods, different being to use is not activated sub pBI101 carrier, and Arabin promotor (Arabin-pro) is inserted the upstream that is fused to gus gene in the pBI101 carrier as Hind III-SalI fragment.Then, be present in the gus gene (CLONTECH in the pBIl01 carrier that is not activated son, Palo Alto, CA, www.clontech.com) had (psr1) (Figure 12 C) of justice or antisense (α-psr1) (Figure 12 D) psrPK gene replaces, produce be subjected to that seed-specific (Arabin) promotor controls justice or antisense construct thing arranged.

Employed Plant Transformation program is described as people such as Katavic (1994).In brief, incubated overnight Agrobacterium tumefaciens strain GV3101, its have help property nopaline plasmid MP90 and contain the psrPK gene constructs and the plant selected marker two advance carrier.Otch site to main or secondary inflorescence branch is come plant tissue is inoculated for three times with the edaphic bacillus cell culture contact that transforms.

Processed plant-growth is to ripe, and the results seed screens (Figure 28) to transformant on selective medium.To determining of transforming be, by southern blotting technique method or polymerase chain reaction technique and adopt psrPK and relevant sequence to determine whether plant contains the gene of transfer to some extent.

The bacterial expression of embodiment 12:psrPK and active definite to protein kinase

In coli expression carrier pGEX (Promega), psrPK protein is expressed.To insert body and increase out from the Bluescript plasmid of the psrPK that contains Arabidopsis, preparation contains the fragment of translation initiation codon, and its previous codon in terminator codon stops (promptly not comprising terminator codon).In adorned pGEX plasmid, it is six extra codons that this plasmid contains 3 ' of the psrPK cDNA that is inserted into terminal Histidine with this fragment cloning.Then, protein is expressed in bacterium, from crude extract psrPK protein is carried out purifying with the pillar of affinity of the residue of the Histidine of seeking and visiting adding.

Adopt the psrPK protein of purifying to carry out the activity that protein kinase is determined in three kinds of different experiments.Referring to Manser et al., (1994); Manser, et al., (1992); Manser, et al., (1995).

Two experiments of front relate to proteinic definite to what growing plants obtained from lacking phosphoric acid salt.With plant tissue homogenate in damping fluid of about 1-5 gram, damping fluid contains 15mM Hepes/KOH pH7.6,40mM KCl, 5mM MgCl ₂, 1mM dithiothreitol (DTT) (DTT), 0.1mM phenmethyl alkylsulfonyl is fluoridized thing (PMSF) (Sigma).Homogenate was placed on ice 30 minutes, and centrifugal 15 minutes then with 13000xg, twice.(CA) described, use Bio-Rad protein detection reagents enriched material is determined proteinic concentration for Bio-Rad Lab., Richmond according to manufacturers.Every kind of protein extract is got 50 micrograms, join that sex change SDS-PAGE goes up or natural gel (Laemmli, 1970) on.For SDS-PAGE, the concentration of the final acrylamide monomer in the thick microgel of 0.75mm (Bio-Rad) is 4% of stacking gel, is 10% of separating gel.Carry out SDS-PAGE with 200V and room temperature, with Coomassie R-250 dyeing (Sigma).For natural gel, it lacks SDS, and the concentration of final acrylamide monomer is 3% in stacking gel, is 7% in separating gel.Gel is walked 1 hour at 4 degrees centigrade in advance with 200V, load sample then, and walk 45 minutes with 200V in 4 degrees centigrade.

Separated protein is by trace (Millipore to the Immobilon P pvdf membrane, Mississauga, Ontario), and, for the sample of SDS sex change, trace is exposed to the denaturing step of the 6M Guanidinium hydrochloride gradient in the MES damping fluid, the MES damping fluid dilution with 50%, 3 hours recovering step is carried out in 5 circulations then with the PBS damping fluid.Then they are exposed to the MES of 25mM, pH6.5 contains the protein and the 25 μ Ci γ of purifying ³²P-ATP, or only contain 25 μ Ci γ ³²P-ATP, the time is 5 minutes, temperature is 22 degrees centigrade, and is following 10 minutes in 4 degrees centigrade again.Then, trace is cleaned, drying to the exograph exposure, is used for testing goal.

For natural gel, trace is exposed to the MES of 25mM, and pH6.5 contains the protein and the 25 μ Ci γ of purifying ³²P-ATP, or only contain 25 μ Ci γ ³²P-ATP, the time is 5 minutes, and temperature is 22 degrees centigrade, is 4 degrees centigrade then, and the time is 10 minutes.Also through cleaning, drying to the exograph exposure, is used for testing goal to these traces.

The 3rd activity experiment uses casein or histone as the artificial substrates (Uesono et al., 1992) for psrPK.The psrPK protein of expressing from the quilt of bacterium is to 20mM Tris-HCl (pH8.0), 10mM MgCl ₂, and 1mM beta-mercaptoethanol dialysis.Isopyknic this mixture is joined 20 μ M ATP, 2 μ M/ml dephosphorylation caseins (or histone) and 25 μ Ci γ ³²Among the P-ATP.According to above-mentioned sample is walked the SDS-Page gel, desiccant gel to the exograph exposure, is used for testing goal.Psr protein makes all phosphorylations of casein and histone.

Embodiment 13: to the psr-Polyglucosidase of the pressure that is in other

The plant culturing of expression analysis

Substantially described according to embodiment 8, make the seed germination and the growth of Arabidopis thaliana (Columbia strain).In investigation during, the concentration of Pi is remained on 5mM to other the replying of environmental stress.To the processing of high salinity, disinfectant NaCl solution is added to the ultimate density of 100mM, this is sublethal concentrations (Saleke et al., 1993).For nonnitrogenous, KNO ₃, and NH ₄NO ₃Substratum, the KCl of volumetric molar concentration such as use to substitute.For the substratum that lacks sulphur, the MgCl of volumetric molar concentration such as use ₂Substitute MgSO ₄The plant that grown 14 days was caused thermal shocking in following 2 hours at 39 degrees centigrade.To containing the flask of 13 age in days plants, be blown into argon gas from the sterile tube that encases with cotton and caused anaerobic condition in 24 hours.In all cases, removed substratum in per 4 days, replace, guarantee not lack the nutritive ingredient that is provided with fresh substratum.Processed plant was gathered in the crops in the time of 14 days, and except the plant of nitrogen is not provided, they,,, cultivated 1 or 3 day behind the additional nutrient through lacking the plant of Pi cultivation in 14 days because there has been serious deficiency disease the 11st day results again.Have only Pi to lack to cause significantly improving to the mRNA level of beta-glucosidase enzyme.

Embodiment 14:RNA trace and genomic library screening

According to Saghai-Maroof, et al. (1984) is described, extracts damping fluid with CTAB genome is extracted from the arabidopsis thaliana material.The genomic dna of about 10 micrograms is with BamHl, EcoRI, Sall, or with the BamHI/SalI Restriction Enzyme at 37 degrees centigrade of enzymolysis that spend the night.After separating enzymolysis product on 0.8% sepharose, (Schleicher and Schuell, Guelph Canada) describedly transfer to dna fragmentation on the Nytran-Plus film according to manufacturers.Use black mustard psr3.1 cDNA to insert body (Malboobi and Lefebvre, 1995) cause the synthetic of probe at random, and guidance subsequently (DuPont NEN, Boston, the hybridization of use on-radiation Du Pont RenaissanceTM test kit MA) according to manufacturers.

Kenton Ko, Queen ' s University, Kingston, Canada provide the genomic library of the environmental Columbia strain of A.Thaliana of being cloned among the EMBL3.Under stringent condition (Malboobi and Lefebvre, 1995), about 200,000 plaque-forming units (pfu) are screened from black mustard psr3.1cDNA clone deutero-probe.Positive colony is by the second time and screening for the third time.Inserting body institute deutero-probe from isolating genomic dna is used in and determines the RNA trace which genomic clone is corresponding to black mustard psr3.1.Be cloned in the selected collection of illustrative plates in Restriction Enzyme site and the subcloning that carries out with routine techniques (Sambrook, et al., 1989) made of genomic clone similar on the trace pattern to black mustard psr3.1 cDNA.

The separation of embodiment 15:RNA and RAN trace

From plant tissue, extract total RNA according to Chirgwin and his colleague described (Chirgwin, et al., 1979).25 micrograms of every kind of RNA extract are loaded into (Sambrook, et al., 1989) on 1% agarose/formaldehyde gel.Carry out the stable laser intensity scanning of RNA trace according to described (Malboobi and Lefebvre, 1995) in the past to radioactive automatic developing.

Embodiment 16: the definite and Computer Analysis of sequence

Preparation is by EcoRI, the resulting dna fragmentation of EcoRI/SalI enzymolysis psr3 genomic clone, and be inserted into pBluescript KS-carrier (Stratagene, LaJolla, CA) in, utilize conventional clone technology (Sambrook, et al., 1989) that these constructions are transformed into suitable intestinal bacteria strain DH5-α (GIBCO BRL, Burlington, Canada) in.Utilize Wizard Megapreps dna purification system (Promega, Madison, WI) preparation plasmid DNA.(United States Biochemical Corp., Cleveland OH) checks order with di-deoxynucleoside acid method with Sequenase Version2 test kit to insert two chains of body.In information storage, used BLASTX program (Gish and State, 1993) to carry out at DNA and protein sequence to the retrieval of homology.To the assembling of encoding sequence and editor and to the analysis of the protein sequence of dna sequence dna and prediction all used suitable LASERGENE software program or Macintosh (DNASTAR, Madison, WI).All cDNA for the psr gene have carried out similar order-checking and homology search.

Embodiment 17: primer extension analysis

Transcription initiation site for beta-glucosidase enzyme is determined with the primer extension analysis method.Oligonucleotide AGCAAAAGCGCCCATGAGAGGAA is being had above-mentioned γ ³²P-dATP exists down and T4 polynucleotide kinase (Promega, Madison, WI) mark.The oligonucleotide that the is labeled MERMAID (Bio/CanScientific of system, Mississauga, Canada) purify and be quenched into the RNA that extracts from the root of Pi hunger, use the anti-phase transcriptase (Pharmacia of AMV then, Baie D ' Urf é Canada) carries out primer extension according to people such as Ausubel described (1995).The result who obtains conventional procedure characterization.

Embodiment 18: proteins extraction and to the gel electrophoresis of beta-glucosidase enzyme

The root tissue of about 10 milligrams of fresh weights contains 15mM HEPES/KOH pH7.6,40mM KCl, 5mMMgCl with the damping fluid homogenate of 500 microlitres in the damping fluid ₂, 1mM dithiothreitol (DTT) (DTT), 0.1mM phenmethyl alkylsulfonyl fluoridize thing (PMSF) (Sigma, St.Louis, MO).Homogenate was placed on ice 30 minutes, then with 13, and centrifugal 15 minutes of 000xg.Removed supernatant liquor also centrifugal 15 minutes.Described (BiO-Rad, Richmond CA) determine according to manufacturers with Bio-Rad protein detection dye formulations enriched material for protein concn in the last supernatant liquor.50 microgram gross proteins carry out electrophoresis (Laemmi, 1970) on the swimming lane of sex change SDS-PAGE or natural gel.To SDS-PAGE, the ultimate density of acrylamide monomer is 4% in the thick microgel of 0.75mm (Bio-Rad), is 10% in stacking gel.Electrophoretic voltage is 200V, and temperature is a room temperature, with Coomassie R-250 dyeing (Sigma).To natural gel, from all reagent, removed SDS, the ultimate density of acrylamide monomer is 3% in stacking gel, is 7% in separating gel.Natural gel moved 30 minutes in advance at 4 degrees centigrade, and voltage is 100V, and temperature is 4 degrees centigrade, adds sample then, in the following 4 degrees centigrade of operations of 200V 3 hours.In order to detect the activity of beta-glucosidase enzyme, natural gel is containing 20mM CaCl ₂PH be balance 15 minutes in 6.5 the 100mM sodium acetate buffer.In same solution, add 0.02% (w/v) Fast Garnet GBC salt (Sigma) and 0.04% (w/v) betanaphthyl β-D-glycopyranoside (Sigma) then, under room temperature, cultivated 3 hours.Equivalent

Those skilled in the art's people will know or just can determine schemes many and the specific embodiments equivalence that this paper introduces by few routine test.These schemes and other equivalence do not break away from the scope of claim regulation of the present invention.

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Claims (71)

1. separated DNA, the functional part of proteinic at least a portion of its encoded light synthesising biological wherein, lacks institute's inductive to translating by phosphoric acid salt of DNA in described biology.

2. separated DNA according to claim 1 wherein further comprises:

(a) nucleotide sequence that intercepts by the quilt of the coded proteinic functional part of SEQ IDNO:1, SEQ ID NO:3 of SEQ ID NO:1, SEQ ID NO:3 or coding; Or

(b) with SEQ ID NO:1, SEQ ID NO:3 the nucleotide sequence of at least 80% homology is arranged, or coding is by the nucleotide sequence that has the quilt of the coded proteinic functional part of the nucleotide sequence of at least 80% homology to intercept with SEQ ID NO:1, SEQ ID NO:3; Or

(c) and the nucleic acid of under moderate stringent condition hybridizing complementary with any (a) or sequence (b).

3. separated nucleic acid, it comprises the 677-1020 nucleotide residue of SEQ ID NO:1.

4. with the DNA of claim 3 50% homology is arranged or under the moderate stringent condition with the DNA and the RNA of its hybridization.

5. by the coded separated polypeptide of the DNA of claim 1.

6. polypeptide according to claim 5, it has the protein kinase activity.

7. separated polypeptide, they are:

(a) by the described dna encoding of claim 2; Or

(b) its minimum part is coded by the 677-1020 nucleotide residue of SEQ ID NO:1.

8. separated nucleic acid, the coding aminoacid sequence that has the active protein of protein kinase and have at least 80% homology, or the aminoacid sequence that has 50% homology with the 190-340 amino-acid residue of SEQID NO:2 with SEQ ID NO:2.

9. Chong Zu expression vector, but comprise the above-mentioned nucleic acid of claim 1 and regulate the sequence place of working and be connected.

10. the cell that contains the described carrier of claim 9.

11. cell according to claim 10, wherein said cell are the photosynthetic organism cells.

12. the transgenic plant that maybe can produce and the plant part that use carrier according to claim 9 to produce, wherein said expression of plants have the protein kinase with the sequence of SEQ ID NO:2 or SEQ ID NO:4 at least 80% homology.

13. the seed of the plant of claim 12.

14. the plant of claim 12 or the tissue culture of plant part.

15. plant according to claim 12 or plant part, wherein said plant are Arabidopsis kind or black mustard (Brassica) kind, or from the plant part of Arabidopsis kind or black mustard (Brassica) kind.

16. transgenosis photosynthetic organism or photosynthetic organism cell, they contain:

(a) DNA of claim 1 or its part; Or

(b) DNA or its part to claim 1 is complementary DNA; Or

(c) above-mentioned (a) or (b) in a kind of, and another or more than one

Different exogenous nucleic acid.

17. a prokaryote contains the described DNA of claim 1 or its part.

18. the offspring of described biology of claim 16 or cell.

19. a method that produces transgenic plant or other photosynthetic organism, wherein said transgenic plant or other photosynthetic organism have growth, breeding or the metabolic characteristics of having been modified, this method comprises:

(a) in the cell or tissue of plant or other photosynthetic organism, introduce exogenous nucleic acid, the protein kinase that this nucleic acid encoding is transcribed under phosphoric acid salt shortage condition in natural species, and its existence in transgenic plant or photosynthetic organism causes growth, breeding or metabolism composition to be modified; And

(b) cell or tissue that will contain exogenous nucleic acid remains under the condition of suitable cell or tissue growth, has growth, breeding or metabolic transgenic plant or other the photosynthetic organism of having been modified thereby produce.

20. method according to claim 19, wherein said transgenic plant or other photosynthetic organism are selected from: angiosperm, gymnosperm, monocotyledons, dicotyledons, non-vascular plant, eucaryon algae and cyanobacteria.

21. method according to claim 19, wherein said nucleic acid are SEQ IDNO:1, SEQ ID NO:3 or their homologue.

22. method according to claim 19, the activity that the phosphoric acid salt of wherein said natural generation lacks the inductive protein kinase is lowered.

23. the transgenic plant that maybe can produce that method according to claim 19 produces.

24. transgenic plant according to claim 23 wherein, are compared with the flowering time and the degree of corresponding natural phant under identical envrionment conditions, flowering time and degree are changed.

25. seed with the transgenic plant that maybe can produce that method produced of claim 19.

26. according to the seed of claim 25, wherein, compare with the phytinic acid composition of the corresponding natural seed of generation under identical envrionment conditions, the phytinic acid composition of seed is lowered.

27. antibody or antibody fragment, it combines with the polypeptide that comprises SEQ ID NO:2, SEQ IDNO:4 or their part.

28. according to the antibody or the antibody fragment of claim 27, wherein said antibody is polyclonal antibody or monoclonal antibody, antibody fragment is the Fab fragment.

29. detection phosphoric acid salt in plant or plant part or the photosynthetic organism at other lacks the method for the expression of inductive protein kinase, this method comprises

(a) cause the shortage of the conspicuous level of its available phosphorus to come the kinase whose expression of induced protein to plant, plant part or other photosynthetic organism;

(b) with plant, plant part or other photosynthetic organism with contact with protein kinase bonded antibody, antibody combined with the epi-position of protein kinase and form antibody: antigenic complex; And

(c) detect antibody: antigenic complex; Wherein, antagonist: the detection of antigenic complex is the index that protein kinase is expressed.

30. according to the separated DNA of claim 1, described DNA comprises

(a) SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or the nucleotide sequence that intercepted, this nucleic acid sequence encoding that is intercepted is by SEQ IDNO:5, the coded proteinic functional part of SEQ ID NO:7, SEQ ID NO:9; Perhaps

(b) nucleotide sequence that has at least 80% homology with SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or the nucleotide sequence that intercepted, this nucleic acid sequence encoding that is intercepted is by having the coded proteinic functional part of nucleotide sequence of at least 80% homology with SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9; Or

(c) with (a) or any sequence complementary (b) and the nucleic acid that can under the moderate stringent condition, hybridize.

31. coded and separated polypeptide by the DNA of claim 30.

32. polypeptide according to claim 31, it has beta-glucosidase activity.

33. a separated nucleic acid, its coding has the protein of beta-glucosidase activity, and has at least 80% sequence homology with SEQ ID NO:5, and perhaps wherein coded protein and SEQ ID NO:6 have at least 50% homology.

34. the carrier of a reorganization, but it contains the nucleic acid of the claim 30 that is connected with adjusting sequence place of working.

35. contain the cell of the described carrier of claim 34.

36. cell according to claim 35, wherein said cell are the cells of photosynthetic organism.

37. with transgenic plant that maybe can produce or the plant part that the described carrier of claim 34 is produced, it expresses the beta-glucosidase enzyme that has at least 80% sequence homology with SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10.

38. the seed of the described plant of claim 37.

39. the tissue culture of described plant of claim 37 or plant part.

40. according to the plant or the plant part of claim 37, plant wherein is Arabidopsis or Brassica, or from Arabidopsis or Brassica deutero-plant part.

41. a prokaryotic cell prokaryocyte, it contains the described DNA of claim 30 or its part.

42. the cell that offspring of the described plant of claim 37 or plant part breed out.

43. a method that produces transgenic plant or other photosynthetic organism, wherein said transgenic plant or other photosynthetic organism have growth, breeding or the metabolism composition characteristics of having been modified, this method comprises:

(a) in the cell or tissue of plant or other photosynthetic organism, introduce exogenous nucleic acid, the beta-glucosidase enzyme that this nucleic acid encoding is transcribed under phosphoric acid salt shortage condition in natural species, and its existence in transgenic plant or photosynthetic organism causes growth, breeding or metabolism to be modified; And

(b) cell or tissue that will contain exogenous nucleic acid remains under the condition of suitable cell or tissue growth;

Thereby produce and to have growth, breeding or metabolic transgenic plant or other the photosynthetic organism of having been modified.

44. according to the described method of claim 43, wherein said transgenic plant or other photosynthetic organism are selected from: vascular plant comprises angiosperm, gymnosperm, monocotyledons and dicotyledons, non-vascular plant, eucaryon algae, and cyanobacteria.

45. according to the described method of claim 43, wherein said nucleic acid is SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:9 or their homologue.

46. according to the described method of claim 43, wherein, the activity that natural phosphate lacks institute's inductive beta-glucosidase enzyme is lowered.

47. the transgenic plant that maybe can produce that produce with the method for claim 43.

48. the seed of the transgenic plant that maybe can produce that produce with the method for claim 43.

49. antibody or antibody fragment, it combines with the polypeptide that comprises SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10 or their part.

50. according to the antibody or the antibody fragment of claim 49, wherein said antibody is polyclonal antibody or monoclonal antibody, described fragment is the Fab fragment.

51. detection phosphoric acid salt in plant or plant part or the photosynthetic organism at other lacks the method for the expression of inductive beta-glucosidase enzyme, this method comprises:

(a) cause the shortage of the conspicuous level of its available phosphorus to induce the expression of beta-glucosidase enzyme to plant, plant part or other photosynthetic organism;

(b) plant, plant part or other photosynthetic organism are contacted with the antibody of claim 49, antibody is combined with the epi-position of beta-glucosidase enzyme and form antibody: antigenic complex; And

(c) detect antibody: antigenic complex;

Wherein, antagonist: the detection of antigenic complex is the index that beta-glucosidase enzyme is expressed.

52. a separated DNA, it comprises

(a) SEQ ID NO:17 or by the nucleotide sequence that intercepted, this nucleic acid sequence encoding that is intercepted is by the coded proteinic functional part of SEQ ID NO:17; Perhaps

(b) nucleotide sequence that has at least 80% homology with SEQ ID NO:17, or the nucleotide sequence that is intercepted, its coding is by having the coded proteinic functional part of nucleotide sequence of at least 80% homology with SEQ ID NO:1 7; Or

(c) with (a) or any sequence complementary (b) and the nucleic acid that can under the moderate stringent condition, hybridize.

53. a separated DNA comprises consecutive nucleotides 1-25,75-115, the 150-187 of SEQ ID NO:17 or 300-316.

54. DNA or RNA, the DNA of itself and claim 53 have 50% homology or under the moderate stringent condition with this DNA hybridization.

55. a separated polypeptide, it is coded by the DNA of claim 52.

56. according to the polypeptide of claim 55, it has phosphoric acid salt transporton activity.

57. a separated polypeptide, it is:

(a) coded by the DNA of claim 52; Perhaps

(b) its at least a portion is nucleotide residue 1-25,75-115, the 150-187 by SEQ ID NO:17, or 300-316 is coded.

58. a separated nucleic acid, its coding have the active protein of phosphoric acid salt transporton and have the aminoacid sequence of at least 80% sequence homology with SEQ ID NO:18.

59. the expression vector of a reorganization, but it contains the nucleic acid of the claim 52 that is connected with adjusting sequence place of working.

60. a cell, it contains the carrier of claim 59.

61. according to the cell of claim 60, wherein said cell is the cell of photosynthetic organism.

62. transgenic plant or plant part, they are had the phosphoric acid salt transporton of at least 80% sequence homology by the carrier generation of using claim 59 and expression and SEQ ID NO:18.

63. the seed of the plant of claim 62.

64. the plant of claim 62 or the tissue culture of plant part.

65. according to the separated DNA of claim 1, it is selected from SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:20, SEQID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27.

66. a nucleotide sequence, any sequence of itself and claim 65 has at least 80% homology.

67. a nucleic acid, any sequence of it and claim 65 is complementary and can hybridize under the moderate stringent condition.

68. a separated polypeptide, it is coded by any nucleotide sequence of claim 65.

69. a method of improving the cold-resistant or anti-frost feature of plant, this method comprises:

(a) introduce the DNA construction in the cell or tissue of plant or other photosynthetic organism, this DNA construction comprises:

(ⅰ) but the constructivity promotor that is connected with the proteinic exogenous nucleic acid of coding psr place of working when described protein is expressed, is lowered with regard to the phosphate content that makes described cell or tissue; Or

(ⅱ) but the cold inductive promotor that is connected with the proteinic exogenous nucleic acid of coding psr place of working when described protein is expressed, is lowered with regard to the phosphate content that makes described cell or tissue; With

(b) described cell or tissue is remained on make under the condition that psr protein expressed;

Thereby produce plant that phosphate content has been lowered or other photosynthetic organism, and improved the cold resistant of plant or other photosynthetic organism or the characteristic of anti-frost.

70. the method by claim 69 produces the plant that maybe can produce.

71. the seed of the plant of claim 70.

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