CN109182350A - Application of the corn Zm675 gene in plant quality improvement - Google Patents

Abstract

The present invention relates to the application of maize Zm675 gene in plant quality improvement. The present invention discloses the application of Zm675 gene, Zm675 protein or expression cassette or vector containing Zm675 gene in increasing plant lysine and/or protein content. The Zm675 gene was obtained by cloning in maize, and the transgenic maize into which the Zm675 gene was introduced was constructed. The lysine content and protein content in the seeds of transgenic maize with enhanced Zm675 gene expression were significantly increased, and the number of protein bodies in maize endosperm cells was significantly increased. At the same time, the excellent traits of high protein and high lysine in the transgenic maize introduced into Zm675 gene can be stably inherited. The Zm675 gene has important application value for increasing the content of lysine and protein in plant grains and improving the nutritional quality of plants.

Description

Application of the corn Zm675 gene in plant quality improvement

Technical field

The present invention relates to biotechnology and genetic breeding fields, specifically, being related to corn Zm675 gene in plant quality Application in improvement.

Background technique

Corn is important cereal crops and forage crop, but protein and certain essential amino acids contain in corn kernel Measure it is lower so that the nutritive value of corn is somewhat limited.Lysine is that first in corn kernel is restricted Amino acid, therefore, the content for improving protein and essential amino acid, especially lysine in seed can improve the nutrition of corn Quality.1964, the discovery of opaque2 Maize mutant opened the beginning of corn nourishment quality-improving research, and O2 gene is compiled The transcription factor of one bZIP class of code, the synthesis of adjustable alcohol soluble protein, and the mutation of O2 gene causes to lack in corn embryosperm The alcohol soluble protein ratio of weary lysine declines, so in endosperm the content ratio of lysine rise (Schmidt et al., 1990).Lysine content 70% -100% (Mertz et higher than conventional corn in the corn embryosperm of opaque-2 (o2) mutant Al., 1964), but since its seed shows as opaque soft endosperm, cause that its low output, percentage of seedgermination are low, seedling is raw Mildew, the not number of drawbacks such as storage endurance and processing quality difference easily occur for long poor, susceptible pest and disease damage.Utilize endosperm modifier The QPM (high-quality protein maize) cultivated by the means of conventional breeding, the shortcomings that overcoming opaque2 mutant, QPM not only relies Histidine content is high, and endosperm hard, and economical character is excellent (Gibbon and Larkins, 2005), has apparent nutrition Advantage and economic use value.But there is also some defects and deficiencies for QPM corn: (1) with conventional corn compared with, QPM corn Yield is lower；(2) QPM production faces serious biological (pest and disease damage) and abiotic (arid, heat, low soil pH, low Soil Nitrogen Deng) constraint.And breeding cycle is long, the feature of low efficiency and predictable difference increases the difficulty for cultivating resistance QPM kind. (3) due to provide QPM character o2 allele be it is recessive, when QPM corn awards normal endosperm zasiokaurin, meeting Lose the excellent inhereditary feature of QPM.Therefore, QPM production needs to be isolated, and which increase the requirement (Liliane to planting environment et al.,2017)。

The development of technique for gene engineering provides new thinking to obtain the corn of high lysine content, on the one hand, passes through Lysine metabolic pathway of synthesizing is adjusted, the content of free lysine in corn kernel can be improved.Reyes etc. is interfered using RNA Technology inhibits the activity of the key enzyme LKR/SDH during lysine catabolic, increases free lysine in corn kernel Content (Reyes et al.2009).But the accumulation of excessive free lysine will affect the development of corn kernel again, in addition Free lysine has unstability, is easy to it in process by destruction (Wenefrida et al., 2013).

On the other hand, by inhibiting the expression of 19-kD or 22-kD alpha-alcohol soluble protein, 19kD or 22kD alcohol in seed is reduced The content of molten albumen can obtain the higher corn of lysine content (Segal et al.2003；Huang et al.2004； Huang et al.2005；) but the seed phenotype of this corn it is the same with o2 mutant, generate farinaceous albumen, producing it Application be restricted (Wu and Messing, 2012).

It is to obtain the corn of high lysine content, and overcome that the qpm (quality protein maize) that will be enriched in lysine is expressed in seed One new approach of the soft problem of endosperm, the key of this method are to obtain the gene money of good high lysine content albumen Source.Currently, mainly a kind of micro-pipe rich in lysine from dicotyledon for corn quality Upgrading combines Protein gene (Chang et al., 2015；Lang et al.,2004；Liu et al.,2015；Yu et al.,2004； Yue J,et al.,2014)。

Summary of the invention

In order to solve the problems in the existing technology, the object of the present invention is to provide corn Zm675 genes in plant product Application in matter improvement.

Inventor has found that the Zm675 gene of corn has the bad ammonia for improving plant in research plant quality improved, process The function of acid content and protein content.The current Unknown Function of corn Zm675 gene, the lysine content for encoding albumen are 18.56%.However, Zm675 gene, other than the albumen of coding has relatively high lysine content, inventor also found raising The expression quantity of Zm675 gene, can not only significantly improve the content of plant lysine, and the content of the protein in plant is also obvious It improves, while the synthesis and accumulation of the proteosome in vegetable seeds also significantly increase.Thus it is speculated that Zm675 gene participates in regulation The formation of proteosome influences the accumulation of other multiple proteins in seed including the protein of high lysine content, into And the effect for improving the lysine and protein content of seed is played, improve the quality of vegetable seeds.

Firstly, the present invention provides Zm675 gene, Zm675 albumen or expression cassette containing Zm675 gene or carrier is improving Application in plant lysine and/or protein content.

Or, Zm675 gene, Zm675 albumen or expression cassette containing Zm675 gene or carrier are in improvement plant quality Using.

Or, Zm675 gene, Zm675 albumen or expression cassette containing Zm675 gene or carrier turn in preparation improvement quality Application in gene plant.

Zm675 gene of the present invention has following nucleotide sequence:

(1) nucleotide sequence as shown in SEQ ID NO.2；

(2) replacement, missing or insertion of the nucleotide sequence as shown in SEQ ID NO.2 through one or more bases obtains Coding have identical function albumen nucleotide sequence；

(3) under strict conditions, can with nucleotide sequence hybridization described in (1) or (2) and coding have identical function The nucleotide sequence of albumen.

Zm675 albumen of the present invention has following amino acid sequence:

(1) amino acid sequence as shown in SEQ ID NO.1；

(2) it replacement of the amino acid sequence as shown in SEQ ID NO.1 through one or more amino acid, missing or is inserted into The amino acid sequence with identical function albumen arrived.

Preferably, application of the present invention is the expression quantity for improving Zm675 gene；

It is highly preferred that the expression quantity for improving Zm675 gene is by Zm675 gene transfered plant.

Zm675 gene source provided by the invention is in corn, and therefore, the expression quantity for improving Zm675 gene can lead to Cross the expression quantity for improving Zm675 gene described in corn or in other plants by described in transgenic method heterogenous expression Zm675 gene.

Plant of the present invention can be any one in unifacial leaf or dicotyledon.

Preferably, plant of the present invention is corn.

Further, the present invention provides a kind of side for preparing the genetically modified plants that lysine and/or protein content improve Method, the method are to improve the expression quantity of Zm675 gene.

Preferably, the expression quantity for improving Zm675 gene is by Zm675 gene transfered plant；The Zm675 gene tool There is following nucleotide sequence:

(1) nucleotide sequence as shown in SEQ ID NO.2；

(2) replacement, missing or insertion of the nucleotide sequence as shown in SEQ ID NO.2 through one or more bases obtains Coding have identical function albumen nucleotide sequence；

(3) under strict conditions, can with nucleotide sequence hybridization described in (1) or (2) and coding have identical function The nucleotide sequence of albumen.

The stringent condition be in 0.1 × SSPE containing 0.1%SDS or 0.1 × SSC solution containing 0.1%SDS, Hybridize at 65 DEG C, and washes film with the solution.

Specifically, the method for preparing the genetically modified plants that lysine and/or protein content improve includes following step It is rapid:

(1) expression vector containing Zm675 gene is constructed；

(2) expression vector is imported into plant, obtains genetically modified plants.

As a kind of preferred embodiment of the invention, the expression vector containing Zm675 gene is based on plant table It is constructed up to carrier pSB130-F128 (ZL201310398102.9)；

The expression vector includes two T-DNA: where T-DNA1 includes by Millet Seed specificity promoter F128 (CN101063139A) the Zm675 expression cassette driven；T-DNA2 includes HPT (the hygromycin phosphoric acid driven by CaMV35S promoter Transferase gene) expression cassette.

It is described by expression vector importing plant be the method infected by Agrobacterium.

The beneficial effects of the present invention are: the present invention clones in corn and obtains Zm675 gene, by improving Zm675 base The expression quantity of cause, constructs transgenic corns.Enhance the content of lysine in the seed of the transgenic corns of Zm675 gene expression It improves 20% or more, up to 41.17%, the content highest of protein improves 12.84%, forms sediment in corn embryosperm cell Powder quantity significantly reduces, and proteosome quantity increased significantly；Meanwhile importing the high protein of the transgenic corns of Zm675 gene It can obtain stablizing heredity with the merit of high-lysine；Moreover, importing the endosperm that Zm675 gene has no effect on corn seed The seed endosperm phenotype of character and germination rate, transgenic corns is normal, and no farinaceous albumen occurs, the germination rate and wild type of seed Without significant difference.Therefore, Zm675 gene can be used for improving lysine and protein content in plant seed, improve the battalion of plant Support quality.

Detailed description of the invention

Fig. 1 is the construction strategy figure of the plant expression vector containing Zm675 gene in embodiment 2.

Fig. 2 is that T0 is detected for the PCR of HPT in the corn regeneration plant of part in embodiment 4.Wherein 3-5 is respectively transgenosis Plant F4, F10 and F11.

Fig. 3 is that T1 is detected for the PCR of Zm675 gene in the plant of part in embodiment 4.Wherein 3-5 is respectively to turn base Because of strain F4, F10 and F11.

Fig. 4 is that R1 is analyzed for the expression of Zm675 gene in corn immature seed in embodiment 6；Using Actin as Reference gene, data are the duplicate average value of biology three times, significance analysis examine according to t (* p < 0.05, * * p < 0.01,***p<0.001)。

Fig. 5 is the transmission electron microscope observing result figure of prematurity corn embryosperm in embodiment 7, wherein Bar=5 μm；PB is represented Proteosome；S represents amylum body.

Fig. 6 is 8 transgenic corn kernel phenotype of embodiment and germination rate, wherein A is seed phenotype, from top to bottom 1st, 3,4,6 row is the seed shot under white light, and the 2nd, 5 row is the corn kernel shot under transmitted light, and the 1st, 2,4,5 row has been Whole seed, the 3rd and 6 rows are the cross section of seed same area；B is seed germination rate, and seed sprouts experiment in triplicate, number According to the average value to test three times, error line is standard deviation.

Specific embodiment

The preferred embodiment of the present invention is described in detail below in conjunction with embodiment.It will be appreciated that following real Providing merely to play the purpose of explanation for example is applied, is not used to limit the scope of the present invention.The skill of this field Art personnel without departing from the spirit and purpose of the present invention, can carry out various modifications and replace to the present invention.

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

The Cloned culturing of 1 Zm675 gene of embodiment

Using corn inbred line B73 as material, blade total serum IgE is extracted, synthesizes cDNA by primed reverse transcription of Oligo dT.

According to maize genomic sequence (maizeGDB:GRMZM2G051675) design primer 675-F1 (SEQ ID NO.3): 5'-CACCAGAGGCGATGTCGTC-3' and 675-R1 (SEQ ID NO.4): 5'-TGGTGA GAGCATCTTCAGTC-3' carries out PCR amplification using cDNA as template.Reaction condition: 95 DEG C, 30sec；58℃,30sec,72 DEG C, 30sec, 30 circulations.Amplified production is connect with carrier T pMD-19T, converts bacillus coli DH 5 alpha, obtains recombinant plasmid.It surveys Sequence the result shows that clone Zm675 coded sequence overall length be 669bp (as shown in SEQ ID NO.2), encode 222 amino acid Residue (as shown in SEQ ID NO.1), coding albumen in lysine content be 18.56% (w/w).

The building of 2 expression vector pSB130-675 of embodiment

Zm675 is cut from above-mentioned carrier T with BamHI and HincII, is expressed with BamHI and Sma I double digestion plant Zm675 segment connect with carrier, is built into recombinant plasmid pSB130- by carrier pSB130-F128 (ZL201310398102.9) 675 (Fig. 1).The plasmid includes two T-DNA.T-DNA1 includes by Millet Seed specificity promoter F128 (CN101063139A) the Zm675 expression cassette driven；T-DNA2 includes HPT (the hygromycin phosphoric acid driven by CaMV35S promoter Transferase gene) expression cassette.After the success of pSB130-675 vector construction, it is transferred in Agrobacterium LBA4404, for corn Genetic transformation.Agrobacterium-mediated Transformation is carried out using conventional method in that art.

The genetic transformation of 3 maize immature embryos of embodiment

The preparation of Agrobacterium bacterium solution: Agrobacterium LBA4404 (pSB130-675) is in YEB solid medium (the 125 μ g/ containing Sm 100 μ g/mL of mL, Kan) on, thallus is collected in 28 DEG C of cultures two days later.With infecting culture medium suspension thalline to OD₆₀₀For 0.6- 0.8,28 DEG C, 75rpm is protected from light shake bacterium 2-4 hours after, the bacterium solution shaken is diluted to OD with culture medium is infected₆₀₀It is standby for 0.3-0.4 With.It is transformation receptor with corn inbred line 178, strips 12 days after the pollination of 178 self-mating systems ratarias (size about 1.5-2.0mm), Culture medium is infected with without Agrobacterium, after impregnating and rinsing 2 times, remaining culture medium is removed, with ready Agrobacterium in advance Bacterium solution infects rataria 15min, and then the rataria infected is placed on aseptic filter paper and is dried up, is transferred in co-culture medium, Upward, 20 DEG C are protected from light co-cultivation 3 days to scultellum.After 3 days, rataria is transferred in recovery media, 28 DEG C after dark culture 7 days, turn Move in the screening and culturing medium containing hygromycin, cultivated under 28 DEG C of dark conditions, every two weeks subculture it is primary (screening conditions be containing The hygromycin of 5mg/L, 15mg/L, 20mg/L, 20mg/L).It is dark by the Transformation of Callus after screening into recovery media Under the conditions of renewal cultivation 7-10 days.Then callus is gone in differential medium, illumination cultivation, the photoperiod is illumination/black It is secretly 16h/8h, subculture is primary every two weeks.Seedling wait differentiate it is long to 3-4cm when, seedling is cut from kanamycin-resistant callus tissue, is moved Enter root induction in root media, when the root system of seedling than it is more developed when be transferred to greenhouse, after hardening in kind to small basin, then It is transferred in greenhouse big basin and continues to cultivate, harvested after seed is mature.Each stage culture medium prescription of corn transformation is shown in Table 1.

Each stage culture medium prescription (1L) of 1 corn transformation of table

The PCR of the genomic DNA of 4 transgenic corns of embodiment is detected

PCR detection: extracting the obtained regeneration plant genomic DNA of embodiment 3, with primer HPT3 (SEQ ID NO.5): 5'-TCGGCTCCAACAATGTCCTG-3' and HPT4 (SEQ ID NO.6): 5'-CGGTCGGCATCTACTCTATTCC-3' into Row PCR amplification detects riddled basins HPT.The purpose band of the amplifiable about 450bp out of positive plant, some materials PCR inspection Survey result such as Fig. 2.

The positive T0 of PCR detection is planted for corn, pollination self obtains T1 for transgenic corns.Due on carrier Two T-DNA independently integrate, have a separation in offspring, therefore whether detect in offspring has Zm675 gene to be transferred to.It is deposited in corn In endogenous Zm675 gene, but endogenous Zm675 gene contains seven intrones, and only wraps in pSB130-675 expression vector Include the CDS of Zm675, thus design across introne primer 675-196-F (SEQ ID NO.7): 5'- ACAAGGCCCACAAGGACG-3' and 675-651-R (SEQ ID NO.8): 5'-CAGGGTCACCACTCAGAAATGA-3' is right Target gene carries out PCR detection.Reaction condition: 95 DEG C, 30sec；56 DEG C, 30sec, 72 DEG C, 30sec, 30 circulations.Primer 675-196-F/675-651-R is across second to the 7th introne, and amplification length is 456bp on CDS, and genomic DNA is Template amplification length is 2286bp；In PCR condition setting by extension of time control on 30sec, genome endogenous sequence because Extension of time is short and cannot be amplified out.As the result is shown, the special sequence of the Zm675 gene of importing can be detected in transgenic line Column, and this band (Fig. 3) is not amplified in wild-type corn genomic DNA.

5 T1 of embodiment is for corn seed lysine and determining the protein quantity

T1 is measured for the lysine content of the seed of transgenic corns using ninhydrin method (Yue J, et al., 2014).15 plants are planted for each transgenic line, 5 plant that wherein target gene PCR detection is positive is chosen and is examined It surveys.The results show that compared with 178 self-mating system of wild-type corn planted simultaneously, relying in the seed of obtained transgenic line Histidine content have it is different degrees of significantly improve, wherein the lysine content increase rate of F4, F10 and F11 strain is respectively 29.41%, 20.58% and 41.17%.Maize seed protein content, nitrogen are measured using semimicro-Kjeldahl method (GB2905-82) Conversion coefficient with protein content is 6.25.Compared with wild type, the protein content of F4, F10 and F11 transgenic line seed There is different degrees of conspicuousness to improve, increase rate is respectively 9.65%, 5.50% and 12.84% (table 2).

Lysine and protein content of 2 T1 of table for transgenic corn seed

Numerical value is average value ± SD, the significance analysis of each transgenic line and WT examine according to t (* p < 0.05, * * p < 0.01,***p<0.001)

Tri- strains of F4, F10 and F11 are carried out with screening and the tracking and monitoring in continuous 5 generation, each transgenic line chooses 5 A plant, the results show that the lysine and protein content of transgenic progeny strain seed are in T₂~T₅Dai Jun has different degrees of mention Height, and stability is preferable.In T5 in seed, the lysine content of F4 and F10 improve 20% or more, and protein content improves 10% Left and right, and the lysine content of F11 is improved up to 40% or more, protein content improves 15% or more (table 3), illustrates Zm675 gene It acts on and stablizing in self-mating system, the merit of high protein and high-lysine can obtain stablizing heredity.

The lysine and protein content increase rate of 3 transgenic corns progeny seed of table

6 T1 of embodiment is analyzed for the expression of Zm675 gene in corn seed

For the raising and the correlation of Zm675 of lysine in verifying F4, F10 and F11 and protein content, transgenosis is had detected The transcriptional level of Zm675 in corn.The T1 of F4, F10 and F11 plant positive for PCR detection are selected, each strain selects 6 Plant extracts 20 days after pollination endosperm RNA, and reverse transcription is at cDNA.Using cDNA as template, with primer 675-196-F/675- 651-R carries out Real-time PCR analysis, and each strain takes the average value of 6 plant.Compared to WT lines, transgenosis The expression quantity of Zm675 is significantly increased (Fig. 4) in strain F4, F10 and F11, wherein the transcriptional level of Zm675 in F11 strain Highest, in conjunction with the embodiments in 5 the lysine content of each transgenic line and protein content analysis as a result, showing each transgenic line The lysine content and protein content difference of system are due to caused by the difference of Zm675 gene expression amount, in a certain range Lysine content and protein content in transgenic corn seed are improved with the raising of Zm675 gene expression amount.

The transmission electron microscope observing of 7 transgenic corns prematurity endosperm of embodiment

T5 is chosen for 20 days after transgenic corns pollination seeds, longitudinal sectional the cutting of about 2mm thickness is cut from endosperm tissue Piece guarantees that the external morphology of institutional framework is not destroyed during slice preparation.Then slice is invaded and is ready in advance Fixer in, the material fixed is subjected to ultra-thin section, piece cut observes under transmission electron microscope.In wild type 178 In the endosperm of self-mating system, amylum body volume is larger, white dispersion shape texture, is distributed more and close；Proteosome is distributed in shallow lake Around powder, gray roundlet is dotted.Compared to wild type, form sediment in tri- transgenic line corn embryosperm cells of F4, F10, F11 Powder quantity significantly reduces, and proteosome quantity increased significantly, and is distributed fine and close (Fig. 5), the results showed that, Zm675 gene participates in adjusting The formation and accumulation for controlling proteosome, influence the product of the multiple proteins in seed including the protein of high lysine content It is tired, and then the effect for improving the lysine and protein content of seed is played, improve the quality of vegetable seeds.

8 Zm675 transgenic corns grain characters of embodiment and germination rate analysis

Take the T of F4, F10 and F11₂For transgenic corn seed and wild-type corn seed, each strain selects three at random Grain under white light and observes each strain Grain Morphology under transmitted light respectively using stereomicroscope.As a result such as the A institute of Fig. 6 Show, compared with wild type, the seed state and no significant difference of transgenic line；Transgenic corns endosperm phenotype is normal, no powder Matter endosperm occurs, and shows that the expression of Zm675 gene does not influence the growth conditions of corn kernel.Meanwhile to F4, F10 and F11 this The corn seed of three transgenic lines has carried out sprouting experiment.Wild type and these three strains of F4, F10 and F11 T are taken respectively₂ It each 100 for transgenic corn seed, sprouts 5 days, observation statistics germination rate.Data analysis shows, the seed of F4, F10 and F11 Germination rate is similar with wild type, in 85% or more (as shown in the B of Fig. 6), illustrates that the expression of Zm675 does not influence corn seed It sprouts.

Bibliography

Chang,Y.,Shen,E.,Wen,L.,Yu,J.,Zhu,D.,and Zhao,Q.(2015).Seed-Specific Expression of the Arabidopsis AtMAP18 gene increases both lysine and total protein content in Maize.PLoS ONE 10,e142952.

Gibbon,B.C.,and Larkins,B.A.(2005).Molecular genetic approaches to developing quality protein maize.Trends Genet.21,227-233.

Huang,S.,Adams,W.R.,Zhou,Q.,Malloy,K.P.,Voyles,D.A.,Anthony,J.,Kriz, A.L.and Luethy,M.H.(2004)Improving nutritional quality of maize proteins by expressing sense and antisense zein genes.J.Agric.Food Chem.52,1958–1964.

Huang,S.,Kruger,D.E.,Frizzi,A.,D'Ordine,R.L.,Florida,C.A.,Adams,W.R., Brown,W.E.,and Luethy,M.H.(2005).High-lysine corn produced by the combination of enhanced lysine biosynthesis and reduced zein accumulation.Plant Biotech J, 2005,3:555-569

Lang,Z.,Zhao,Q.,Yu,J.,Zhu,D.,and Ao.,G.(2004).Cloning of potato SBgLR gene and its intron splicing in transgenic maize.Plant Sci.166,1227-1233.

Liliane N.T.,Charles S.M.,Eddy L.M.N.,NoéW.,(2017)Breeding for Quality Protein Maize(QPM)Varieties:A Review Agronomy.7,80；doi:10.3390/ agronomy7040080

Liu,C.,Li,S.,Yue,J.,Xiao,W.,Zhao,Q.,Zhu,D.,Yu,J.(2015).Microtubule- associated protein SBgLR facilitates storage protein deposition and its expression leads to lysine content increase in transgenic maize endosperm.Int.J.Mol.Sci.16,29772-29786.

Mertz,E.T.,Bates,L.S.,and Nelson,O.E.(1964).Mutant gene that changes protein composition and increases lysine content of maize endosperm.Science 145,279-280.

Reyes,A.R.,Bonin C.P.,Houmard N.M.,Huang S.and Malvar T.M.(2009) .Genetic manipulation of lysine catabolism in maize kernels.Plant Mol Biol,69 (1):81-89

Schmidt,R.J.,Burr,F.A.,Aukerman,M.J.,and Burr,B.(1990).Maize regulatory gene opaque-2encodes a protein with a″leucine-zipper″motif that binds to zein DNA.Proc.Natl.Acad.Sci.U.S.A.87,46-50.

Segal,G.,Song,R.,and Messing,J.(2003).A new opaque variant of maize by a single dominant RNA-interference-inducing transgene.Genetics 165,387- 397.

Wenefrida,I.,Utomo,H.S.,and Linscombe,S.D.(2013).Mutational breeding and genetic engineering in the development of high grain protein content.J.Agric.Food Chem.61,11702-11710.

Wu,Y.,and Messing,J.(2012).RNA interference can rebalance the nitrogen sink of maize seeds without losing hard endosperm.PLoS ONE 7,e32850.

Yu,J.,Peng,P.,Zhang,X.,Zhao,Q.,Zhu,D.,Sun,X.,Liu,J.,and Ao,G.(2005) .Seed-specific expression of the lysine-rich protein gene sb401 significantly increases both lysine and total protein content in maize seeds.Food Nutr.Bull.26,427-431.

Yue J,Li C,Zhao Q,Zhu D,Yu J.Seed-Specific Expression of a Lysine- Rich Protein Gene,GhLRP,from Cotton Significantly Increases the Lysine Content in Maize Seeds.International Journal of Molecular Sciences 2014；15 (4):5350–5365.

The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, without departing from the technical principles of the invention, several improvements and modifications can also be made, these improvements and modifications Also it should be regarded as protection scope of the present invention.

Sequence table

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Claims (10)

1.Zm675 gene, Zm675 albumen or expression cassette containing Zm675 gene or carrier are improving plant lysine and/or egg Application in white matter content.

2.Zm675 gene, Zm675 albumen or the application of expression cassette or carrier in improvement plant quality containing Zm675 gene.

The transgenosis of 3.Zm675 gene, Zm675 albumen or expression cassette containing Zm675 gene or carrier in preparation improvement quality Application in plant.

4. described in any item applications according to claim 1~3, which is characterized in that the Zm675 gene has following nucleosides Acid sequence:

(1) nucleotide sequence as shown in SEQ ID NO.2；

(2) volume that replacement, missing or insertion of the nucleotide sequence as shown in SEQ ID NO.2 through one or more bases obtain Code has the nucleotide sequence of identical function albumen；

(3) under strict conditions, can with nucleotide sequence hybridization described in (1) or (2) and coding have identical function albumen Nucleotide sequence.

5. described in any item applications according to claim 1~3, which is characterized in that the Zm675 albumen has following amino Acid sequence:

(1) amino acid sequence as shown in SEQ ID NO.1；

(2) replacement, missing or insertion of the amino acid sequence as shown in SEQ ID NO.1 through one or more amino acid obtain Amino acid sequence with identical function albumen.

6. described in any item applications according to claim 1~5, which is characterized in that the application is the table for improving Zm675 gene Up to amount；Preferably, the expression quantity for improving Zm675 gene is by Zm675 gene transfered plant.

7. described in any item applications according to claim 1~6, which is characterized in that the plant is unifacial leaf or dicotyledonous plant Object.

8. a kind of method for preparing the genetically modified plants that lysine and/or protein content improve, which is characterized in that the method For the expression quantity for improving Zm675 gene.

9. according to the method described in claim 8, it is characterized in that, the expression quantity for improving Zm675 gene is by Zm675 base Because importing plant；

The Zm675 gene has following nucleotide sequence:

(1) nucleotide sequence as shown in SEQ ID NO.2；

(2) volume that replacement, missing or insertion of the nucleotide sequence as shown in SEQ ID NO.2 through one or more bases obtain Code has the nucleotide sequence of identical function albumen；

(3) under strict conditions, can with nucleotide sequence hybridization described in (1) or (2) and coding have identical function albumen Nucleotide sequence.

10. method according to claim 8 or claim 9, which comprises the steps of:

(1) expression vector containing Zm675 gene is constructed；

(2) expression vector is imported into plant, obtains genetically modified plants.