CN101058816A - Rice OsCS coded sequence and application thereof - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, and specifically relates to a coding sequence of citrate synthase expressed in rice and its application in improving plant citric acid secretion, and is expected to correspondingly improve the aluminum toxicity resistance of plants. The present invention relates to a recombinant expression vector containing the above gene, and also relates to said expression vector transformed plant cells, and said gene transgenic plants produced by said transformed cells. The invention expresses the gene in the plant, and the citric acid secretion of the obtained transgenic plant is significantly increased, and the resistance to aluminum toxicity is also correspondingly enhanced.

Description

Rice OsCS coding sequence and its application

technical field

The invention belongs to the technical fields of molecular biology, physiology and genetic engineering, in particular to the cloning, copy number analysis and gene expression pattern analysis of a citrate synthase (rice citrate synthase, OsCS) expressed in rice , the construction of transgenic vectors, plant transformation, the acquisition of transgenic plants and the identification of corresponding physiological and biochemical characteristics.

Background technique

Nearly 40% of the world's cultivated soil is eroded by acid rain. Among them, aluminum (Al) toxicity is a key factor limiting crop yield in acidic soils. The use of genetic engineering to breed good varieties adapted to poor soil has a certain application potential. The organic acids secreted by roots are of certain importance to the absorption of mineral nutrients by plants. Most importantly, the secretion of organic acids improves the resistance of plants to aluminum toxicity, mainly because organic acids can chelate aluminum ions in the root zone, reducing the amount of aluminum entering cells, thus protecting plants from aluminum toxicity. Organic acids are closely related to resistance to aluminum toxicity, and have been reported in species such as Arabidopsis thaliana, oats and wheat. After aluminum poisoning treatment, different plants and different varieties of the same plant have different types and abilities to secrete organic acids, mainly including citric acid, malic acid, succinic acid and oxalic acid.

Due to the effectiveness of citric acid in anti-aluminum toxicity, citrate synthase has become a hot research topic. Citrate synthase is an enzyme in the initiation reaction of the tricarboxylic acid cycle in the mitochondria and the glyoxylate cycle in the plastid, which catalyzes the production of citrate from CoA and oxaloacetate. The generated citric acid not only serves as an intermediate product of metabolism, but also has the function of chelating toxin ions secreted from the plant body. Therefore, the gene cloning and transgenic work of citrate synthase, the key enzyme of citric acid synthesis, is also particularly important. At present, genes encoding citrate synthase have been isolated in plant species such as Arabidopsis thaliana and carrot, and most of them are mitochondria-specific expression. The genetic transformation function of the citric acid synthase gene proves that the expression of the gene can increase the citric acid secretion of the transgenic plant and the aluminum toxicity resistance of the plant.

The present invention studies a technique of cloning citric acid synthase (OsCS) which catalyzes the generation of citric acid from rice, and improving the excretion of citric acid after being transferred into tobacco. The application of this technology can not only increase the amount of citric acid excretion in plants under aluminum poisoning treatment, but it is expected that plants can grow normally in a soil environment with high aluminum content, and at the same time, it may increase the effective utilization of phosphorus, and can expand plants (such as rice) and so on. cultivation range.

Before the publication of the present invention, the rice OsCS sequence and its nucleic acid sequence mentioned in this patent application have not been disclosed or reported.

Contents of the invention

The first object of the present invention is to provide a new rice gene OsCS, which is a mitochondrial citrate synthase gene.

The second object of the present invention is to provide the application of the rice protein OsCS coding sequence in improving plant citric acid secretion and aluminum toxicity resistance by transgenic technology.

One aspect of the present invention provides an isolated DNA molecule encoding the open reading frame of rice citrate synthase, specifically referring to the nucleotide sequence of SEQ ID NO.1.

Another aspect of the present invention also provides the polypeptide encoded by the above sequence having the amino acid sequence shown in SEQ ID NO.2.

The present invention also provides a method for detecting the spatiotemporal expression pattern of OsCS mRNA, the steps of which are as follows:

(1) The leaves and roots of rice seedlings (two leaves and one heart stage) were taken, fixed, and sectioned in paraffin.

(2) Use the RNA of the open reading frame of OsCS as a probe, and hybridize to develop color.

The present invention also provides a method for utilizing transgenic technology to transform the nucleotide sequence encoding rice OsCS enzyme activity into tobacco to increase the amount of citric acid secretion, the steps are as follows:

(1) connecting the open reading frame of the rice OsCS gene to the plant expression vector to form a plant expression vector containing the rice OsCS gene (nucleotide sequence of SEQID NO.1);

(2) Transform the expression vector in step (1) into Agrobacterium, co-cultivate the Agrobacterium containing the expression vector with tobacco leaves, and culture in dark for 2 days at 25±2°C;

(3) Through antibiotic screening and PCR identification, the transformed cells of the rice OsCS gene are obtained and the transgenic plants are regenerated. The citric acid secretion of the transgenic plants containing the rice OsCS gene was increased to a greater extent.

The present invention also provides a method for detecting the copy number of rice citrate synthase in a sample, which comprises using the sequence shown in SEQ ID NO.1 to hybridize with the sample, and then detecting whether the probe is combined. The sample is rice and transgenic tobacco genome DNA, the nucleotide sequence after restriction endonuclease digestion.

The probe described here is the following nucleic acid molecule, which comprises the reverse complementary sequence of the 893-910th continuous nucleotides and the 1405-1425th continuous nucleotides in the DNA molecule shown in SEQ ID NO.1.

The present invention also provides a method for detecting the expression pattern of rice citrate synthase in a sample, which includes preparing a probe with the sequence shown in SEQ ID NO.1, hybridizing with the sample, and then detecting whether the combination occurs. The samples were paraffin sections of leaves and roots of rice seedlings.

The present invention also provides a nucleic acid molecule for identifying transgenic tobacco, which comprises performing PCR reaction with a sample using the aforementioned nucleotide sequence, and then detecting whether a target segment is amplified; the sample is genomic DNA of transgenic tobacco.

Another aspect of the present invention also provides a method for measuring the excretion of transgenic tobacco citric acid, the steps of which are as follows:

(1) After taking the extracellular part (culture solution) and passing through the anion and cation exchange resin in sequence, the organic acid retained on the anion exchange resin was eluted and collected with hydrochloric acid, and the eluate was concentrated with a rotary evaporator, dissolved in 700 μl of water, and carried out Determination by high performance liquid chromatography.

(2) Measuring procedure of high-performance liquid chromatography: the mobile phase is 50 mM HClO ₄ , the column temperature is 50° C., the flow rate is 1.0 ml/min, and the measurement takes 20 min.

The full-length nucleotide sequence of the rice OsCS protein of the present invention or its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequence, and cDNA libraries prepared by commercially available cDNA or conventional methods known to those skilled in the art can be used As a template, related sequences are amplified. When the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then splice together the amplified fragments in the correct order.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This usually involves cloning it into a vector, transforming it into cells, and then isolating relevant sequences from the proliferated host cells by conventional methods.

Table 1 is that the protein sequence encoded by the rice OsCS gene of the present invention is the same as that of carrot (Daucus carota), tobacco (Nicotiana tabacum), sugar beet (Beta vulgaris subsp.), Arabidopsis thaliana, and orange (Citrusjunos) amino acid sequence Origin comparison table (identical amino acid sequences are represented by "*"; relatively conserved sequences are represented by ":"; mitochondrial target signals are represented by "bold and underlined").

Description of drawings

Figure 1 Southern blot identification of rice OsCS gene. Among them, 1-3 are 5 μg of genomic DNA digested by DraI, HindIII and EcoR I respectively. The right side of the picture is the λ-EcoT14 I digest marker.

Fig.2 In situ hybridization results of roots and leaves of OsCS under normal and Al treatment, where A, B, C, D are roots, E, F, G, H are leaves, A, E are Al treatment for 1 day, B, F are Al treatment for 3 days, C, G for Al treatment for 5 days, D, H for the control.

Figure 3 PCR identification of transgenic plants. Among them, EV30 is the tobacco plant transfected with empty vector; CS8, CS42, CS50 are the tobacco plants positively transfected with exogenous OsCS. A, detection of specific primers; B, detection of carrier primers and specific primers.

Figure 4 Southern blot identification of transgenic plants. Among them, EV30 is the tobacco plant transfected with empty vector; CS8, CS42, CS50 are the tobacco plants positively transfected with exogenous OsCS.

Figure 5 Western blot identification of transgenic plants. Among them, EV30 is the tobacco plant transfected with empty vector; CS8, CS42, CS50 are the tobacco plants positively transfected with exogenous OsCS.

Fig. 6 Determination of root length of transgenic plants. Among them, EV30 is the tobacco plant transfected with empty vector; CS8, CS42, CS50 are the tobacco plants positively transfected with exogenous OsCS. Al is 0.5mM CaCl ₂ -50μM AlCl3 (pH4.5) treatment 1d to measure the root length, 0.5mM _CaCl2 (pH4.5) treatment as the control CK.

Figure 7 High performance liquid chromatography (HPLC) identification of transgenic plants. Among them, EV30 is the tobacco transformed with empty vector; CS8, CS42, CS50 are the tobacco plants transformed with exogenous OsCS forward. 0.5mM CaCl ₂ -50μM AlCl3 (pH4.5) treated 1d.

Fig. 8 Detection of growth of transgenic plants treated with aluminum. Among them, EV30 is the tobacco transfected with empty vector; CS42 is the tobacco T1 plant transfected with exogenous OsCS.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples is usually according to conventional conditions, such as molecular cloning such as Sambrook: the conditions described in the experimental manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's suggestion conditions of.

Example 1

Cloning of Rice OsCS Gene

1. Take the rice leaves grown in the greenhouse (25°C) (at the stage of two leaves and one heart) and immediately put them in liquid nitrogen for cryopreservation. It should be noted that the materials selected in this experiment are not necessarily related to the selected rice varieties.

2. Extraction of DNA

Take part of the tissue, grind it with a mortar, add it to a 50ml centrifuge tube filled with preheated lysate, keep it warm at 65°C for 40min, and centrifuge. RNaseA (0.5 μg/ml) was added to the supernatant, and the RNA was digested at 65°C for 1 h; the protein was pumped once with an equal volume of phenol/chloroform/isoamyl alcohol (25/24/1), the DNA was precipitated with ethanol, and washed twice with 70% ethanol. Repeat, and finally dissolve the DNA with TE solution. 0.8% agarose gel was used to check the quality of DNA.

3. Cloning of the full-length gene

The degenerate primers were designed according to the CS homologous sequences of other plants, and a band of about 1.4kb was amplified from rice cDNA by RT-PCR. The PCR product was recovered, connected to a T-vector, and sequenced using SP6 and T7 as universal primers. The sequencing results were submitted to the NCBI non-redundant database, and the BLAST results showed that this sequence and the corresponding citrate synthase sequences in other species were highly conserved.

Example 2

Sequence information and homology analysis of rice OsCS gene

The length of the novel rice OsCS gene of the present invention is 1425bp, and the detailed sequence is shown in SEQ ID NO.1. The polypeptide encoded by the gene consists of 475 amino acid residues, with a molecular weight of 52443.55 Daltons and an isoelectric point of 8.268. See SEQ ID NO.2 for the detailed sequence.

The coding region sequence of the rice OsCS full-length gene and its encoded protein sequence were analyzed by the BLAST program in the Non-redundant GeneBank+EMBL+DDBJ+PDB and Non-redundant GeneBank CDS translations+PDB+SwissPort+Superdate+PIR databases. The homology of acid and protein was detected, and it was found that it has certain homology with the CS of carrot, tobacco, sugar beet, Arabidopsis and citrus. At the amino acid level, there is greater than 70% similarity (see Table 1). It can be seen from the above that the OsCS gene has high homology with the CS genes of carrot, tobacco, sugar beet, Arabidopsis, and citrus, and it can be considered that there is also a high similarity in function.

The result of BLAST indicated that the gene obtained from rice might be citrate synthase gene. The known citrate synthase gene catalyzes the synthesis of citric acid in the TCA cycle. Transferring it into plants can increase the secretion of citric acid in plants and at the same time improve the resistance to aluminum toxicity, so it is speculated that this gene has the same function.

Example 3

Copy number analysis of rice OsCS gene

A large amount of rice genomic DNA was extracted by CTAB method, and 10 μg of DNA was digested with DraI, EcoRI and HindIII respectively, and the fragments were separated by 0.8% agarose gel, and the DNA was transferred to Hybond-N ⁺ nylon membrane and fixed; The OsCS gene was used as a probe for Southern hybridization to identify its copy number in rice. The results showed that the OsCS gene had multiple copies.

Example 4

Expression pattern analysis of rice OsCS

The spatial and temporal expression pattern of rice OsCS was detected by in situ hybridization. After the aluminum treatment, the leaves and roots of rice seedlings (two-leaf and one-heart stage) were taken respectively, sectioned in paraffin, hybridized, and developed for color development. The results showed that under normal culture conditions, OsCS was expressed in both leaves and roots of rice, and the expression intensity in leaves was higher than that in roots. The effect of Al treatment on the expression of OsCS was mainly manifested in the roots. With the prolongation of treatment time, the expression of OsCS was slightly increased.

Example 5

Eukaryotic expression of rice OsCS gene in tobacco cells and phenotypic identification of transgenic plants

Construction of expression vector containing target gene (rice OsCS gene)

Design degenerate primers according to other plant CS homologous sequences, design primers that amplify the complete coding reading frame, and introduce restriction endonuclease recognition sites (depending on the selected vector) on the forward and reverse primers respectively, so that Construction of expression vectors. Using the amplified product obtained in Example 1 as a template, after PCR amplification, the rice OsCS gene was forward cloned into the binary expression vector pBI121, and transformed into Agrobacterium under the premise of ensuring the reading frame. Transformation of the model plant Nicotiana tabacum using the leaf disc method.

Tobacco Transformation Using the Leaf Disk Method

1. Use a sterilized toothpick to pick the positive clones on the YEB selection medium, inoculate them in 2ml of YEB liquid (rifampicin 40mg/L, streptomycin 50mg/L, kanamycin 50mg/L), and culture with shaking at 28°C 200rpm 24-36h;

2. Centrifuge at 4000g for 10min at room temperature;

3. Discard the supernatant, resuspend the bacteria with 1/2 MS liquid medium, and dilute to 5-20 times the original volume, so that OD ₆₀₀ = about 0.5;

4. Take the aseptic leaves of tobacco that have grown for about two weeks, remove the main veins of the leaves, and cut them into small leaves of about 1 square centimeter;

5. Put the leaves into the prepared bacterial solution, soak for 2-5 minutes, and blot the bacterial solution on sterile filter paper;

6. Put the infected leaves on MS medium and culture in dark at 28°C for 48h;

7. Transfer the leaves to the callus medium (MS+6-BA 1.0mg/L+NAA 0.1mg/L+Kan50mg/L+cef250mg/L), culture under light at 25-28°C, see in 7-15 days callus formation;

8. Differentiated buds can be seen to grow after about 20 days. After the buds grow up, cut them off and place them on the rooting medium (1/2MS+NAA(0.5mg/L+Kan50mg/L+cef250mg/L);

9. After the root system develops, take out the plant, wash the attached solid medium with sterile water, move it into the soil, and cultivate it in the greenhouse.

Molecular characterization of transgenic plants

A small amount of genomic DNA of transgenic plants was extracted by CTAB method, and the tobacco transfected with pBI121 empty vector was used as a negative control. Primers were designed according to the specific sequence of rice OsCS gene, and PCR detection was carried out (partial results).

Transgenic plants for phenotypic identification

A number of transgenic plants were obtained, among which the citric acid secretion of three transgenic tobacco increased by about 100%, 60% and 200% respectively compared with the empty-carrying type. It was speculated that the overexpression of exogenous OsCS gene in host tobacco increased the secretion of tobacco citric acid. The test results show that the rice OsCS gene obtained in Example 1 is a functional gene, which can be used in the research and industrialization of improving the aluminum toxicity resistance of rice using transgenic technology.

Table 1

Comparison of amino acid sequence homology between rice OsCS gene encoded protein sequence and carrot (Daucus carota), tobacco (Nicotiana tabacum), sugar beet (Beta vulgaris subsp.), Arabidopsis thaliana, citrus (Citrus junos)

V

LL

L

AVQQSNLSNTVRWFQVQTSASDLDLRSQLKELIPEQQERIKKLK 60Daucus carota MVFF

V

LL

L

AVQQSNLSNTVRWFQVQTSASDLDLRSQLKELIPEQQERIKKLK 60

V

LL

L

AVQQTNLSNSVRWLQVQTSSG-LDLRSELQELIPEQQDRLKKLK 59Nicotiana tabacum MVFY

V

LL

L

AVQQTNLSNSVRWLQVQTSSG-LDLRSELQELIPEQQDRLKKLK 59

Beta vulgaris subsp. -------------------------------------SSNLDLRSELQELIPEQQERLKKIK 25

V

AF L

VGQQSSLSNSVRWIQMQ-SSTDLDLKSQLQELIPEOQDRLKKLK 59Arabidopsis thaliana MVFF

V

AF L

VGQQSSLSNSVRWIQMQ-SSTDLDLKSQLQELIPEOQDRLKKLK 59

V

AL

L

VGQQSNLSNSVRWLQMQ-SSSDLDLHSOLKEMIPE00ERLKKVK 59Citrus junos MAFF

V

AL

L

VGQQSNLSNSVRWLQMQ-SSSDDLDLHSOLKEMIPE00ERLKKVK 59

L

AV

L

VAQEATTLGGVRWLQMQ-SASDLDLKSQLQELIPEQQDRLKKLK 59OsCS MAFF

L

AV

L

VAQEATTLGGVRWLQMQ-SASDLDLKSQLQELIPEQQDRLKKLK 59

                : ***:*:*:*:*****:*:**:*

Daucus carota AEHGKVQLGNITVDMVLGGMRGMTGLLWETSLLDPEEGIRFRGLSIPECQKLLPGAKPGG 120

Nicotiana tabacum SEHGKVQLGNITVDMVLGGMRGMTGLLWETSLLDPDEGIRFRGLSIYECQKVLPAAKPGG 119

Beta vulgaris subsp. KEFGSFQLGNINVDMVLGGMRGMTGLLWETSLLDPEEGIRFRGFSIPECQKLLPAASAGA 85

Arabidopsis thaliana SEHGKVQLGNITVDMVIGGMRGMTGLLWETSLLDPEEGIRFRGLSIPECQKVLPTAQSGA 119

Citrus junos SELGKAQLGNITVDMVIGGMRGMTGLLWETSLLDPDEGIRFRGLSIPECQKLLPAAKPDG 119

OsCS SEHGKVQLGNITVDMVLGGMRGMTGMLWETSLLDPDEGIRFRGLSIPERQKVLPTAVKDG 119

* *. *****.****:********:********:******:** * **:** * ..

Daucus carota EPLPEGLLWLLLTGKVPTKEQVDALSAELRSRAAVPEHVYKTIDALPVTAHPMTQFATGV 180

Nicotiana tabacum EPLPEGLLWLLLTGKVPSKEQVDSLSQELRSRATVPDHVYKTIDALPVTAHPMTQFATGV 179

Beta vulgaris subsp. EPLPEGLLWLLLTGKVPSKEQVDALSADLRKRASIPDHVYKTIDALPITAHPMTQFCTGV 145

Arabidopsis thaliana EPLPEGLLWLLLTGKVPSKEQVEALSKDLANRAAVPDYVYNAIDALPSTAHPMTQFASGV 179

Citrus junos EPLPEGLLWLLLTGKVPSKEQVDGLSKELRDRATVPDYVYKAIDALPVTAHPMTQFASGV 179

OsCS EPLPEGLLWLLLTGKVPTKEQVDALSKELASSRSSVPGHVYEAIDALPVTAHPMTQFTTGV 179

*****************:****:.** :* .*:::* :**::************ ** :**

Daucus carota MALQVQSEFQKA-YEKGIHKTKYWEPTYEDSITLIAQLP-VVAAYIYRRMYKNGQSISTD 238

Nicotiana tabacum MALQVQSEFQKA-YEKGIHKSKLWEPTYEDSMSLIAQVP-LVAAYVYRRMYKNGNTIPKD 237

Beta vulgaris subsp. MALQTQSEFQKA-YEKGIHKSKFWEPTYEDCLSLIAQVP-VVAAYVYRRMYKNGQVIPLD 203

Arabidopsis thaliana MALQVQSEFQKA-YENGIHKSKFWEPTYEDCLNLIARVP-VVAAYVYRRMYKNGDSIPSD 237

Citrus junos MALQVQSEFQEA-YEKGIHKSKYWEPTYEDSLNLIARVP-VVAAYVYQRIYKDGKIIPKD 237

OsCS MALQVESEFQKSPMTKGMSKSKFWEPTYERLLKFDSPSSSGLSYVYRRIFKGGKTIAAD 239

****..****:: :*: *:* **** :.: : . :*:*:*::*.*.*.*

Daucus carota DSLDYGANFAHMLGYDSPSMQELMRLYVTIHTDHEGGNVSAHTGHLVASALSDPYLSFAA 298

Nicotiana tabacum DSLDYGANFAHMLGFSSSDMHELMKLYVTIHSDHEGGNVSAHTGHLVASALSDPYLSFAA 297

Beta vulgaris subsp. DSLDYGGNFAHMLGFDSPQMLELMRLYVTIHSDHEGGNVSAHTGHLVGSPLSDPYLSFAA 263

Arabidopsis thaliana KSLDYGANFSHMLGFDDEKVKELMRLYITIHSDHEGGNVSAHTGHLVGSALSDPYLSFAA 297

Citrus junos DSLDYGGNFSHMLGFDDPKMLELMRLYVTIHSDHEGGNVSAHTGHLVASALSDPYLSFAA 297

OsCS NALDYAANFSHMLGFDDPKMLELMRLYITIHTDHEGGNVSAHTGHLVGSALSDPYLSFAA 299

.:***..**:****:.. .: ***:**:***:**************.*.** ***********

Daucus carota ALNGLAGPLHGLANQEVLLWIKSVVSECGENVTKEQLKDYIWKTLNSGKVVPGYGHGVLR 358

Nicotiana tabacum ALNGLAGPLHGLANQEVLLWIKSVVEECGENISKEQLKDYAWKTLKSGKVVPGFGHGVLR 357

Beta vulgaris subsp. ALNGLAGPLHGLANQEVLLWIKSVVDECGENISTEQLKDYVWKTLNSGKVVPGFGLGVLR 323

Arabidopsis thaliana ALNGLAGPLHGLANQEVLLWIKSVVEECGEDISKEQLKEYVWKTLNSGKVIPGYGHGVLR 357

Citrus junos ALNGLAGPLHGLANQEVLLWIKSVVDECGENVTTEQLKDYVWKTLNSGKVVPGFGHGVLR 357

OsCS ALNGLAGPLHGLANQEVLLWIKSVIGETGSDVTTDQLKEYVWKTLKGGKVVPGFGHGVLR 359

*************************: * *.:::.:***:* ****:.***:** :* ****

Daucus carota NTDPRYICQREFALKHLPDDPLFQLVSNLFEVVPPILTELGKVKNPWPNVDAHSGVLLNH 418

Nicotiana tabacum KTDPRYTCQREFALKHLPEDPLFQLVAKLYEVFLQFLQNLAKLN-PWPNVDAHSGVLLNY 416

Beta vulgaris subsp. KTDPRYTCQREFALKHLPDDPFFQLVSKLYEVVPPILLELGKVKNPWPNVDAHSGVLLNH 383

Arabidopsis thaliana NTDPRYVCQREFALKHLPDDPLFQLVSKLYEVVPPVLTELGKVKNPWPNVDAHSGVLLNH 417

Citrus junos KTDPRYTCQREFALKHLPDDPLFQLVSKLFEVVPPILTKLGKVKNPWPNVDAHSGVLLNH 417

OsCS KTDPRYTCQREFALKYLPEDPLFQLVSKLYEVVPPILTELGKVKNPWPNVDAHSGVLLNH 419

:*************:**:**:****::*:**. .*:*.*::********** ****:

Daucus carota YGLTEARYYTVLFGVSRAIGICSQLVWDRALGLPLERPKSVTMEWLENHCKKSS--472

Nicotiana tabacum YGLTEARYYTVLFGVSRALGICSQLIWDRALGLPLERPKSVTMEWLENHCKKA---469

Beta vulgaris subsp. YGLTEARYYTVLFGVSRSLGICSQLIWDRALGLPLERPKSVTMEWLEKFCKRRA--437

Arabidopsis thaliana YGLTEARYYTVLFGVSRSLGICSQLIWDRA

LGLALERPKSVTMDWLEAHCKKASSA 473

Citrus junos FGLAEARYYTVLFGVSRSLGICSQLIWDRALGLPLERPKSVTLDWIE--------- 464

OsCS FGLSEARYYTVLFGVRSSIGIGSQLIWDRALGLPLERPKSVTMEWLENHCKKVAA-474

    : **:**************::** ***:********.********::*:*

The sequences and symbols involved in the present invention are listed as follows:

(1) Information on SEQ ID NO.1

(i) Sequential features:

(A) Length: 1425bp

(B) Type: Nucleotide

(C) Chain type: single chain-

(D) Topology: Linear

(ii) Molecule type: Nucleic acid

(iii) Sequence description: SEQ ID NO.1

1 ATGGCGTTCTTCAGGGgCCTGACCGCGGTGTCGAGGCTTCGATCCCGCGTGGCACAGGAG

61 GCCACCACGCTTGGTGGTGTGCGATGGCTGCAGATGCAGAGCGCATCTGATCTTGATCTC

121 AAGTCCCAGCTGCAGGAATTGATTCCTGAACAACAGGACCGCTTAAAGAAACTTAAATCG

181 GAGCATGGAAAGGTCCAACTTGGAAATATAACAGTCGATATGGTCCTTGGTGGGATGAGA

241 GGGATGACTGGAATGCTTTGGGAAACATCATTGCTTGACCCGGATGAGGGTATTCGTTTT

301 AGGGGTCTCTCGATTCCAGAGCGCCAGAAAGTGCTGCCGACAGCAGTTAAAGATGGGGAG

361 CCTTTGCCTGAGGGTCTACTTTGGCTTCTTTTGACCGGAAAGGTGCCAACCAAAGAGCAA

421 GTTGATGCTCTATCAAAGGAATTGGCTAGTCGTTCGAGTGTTCCAGGTCATGTGTATGAG

481 GCAATCGATGCTCTTCCTGTAACTGCTCATCCGATGACCCAGTTTACCACAGGAGTGATG

541 GCACTTCAGGTGGAGAGTGAGTTTCAAAAAAAGCCCTATGACAAAGGGAATGTCAAAAATCA

601 AAGTTCTGGGAGCCTACCTATGAAAGATTGCTTAAATTTGATAGCTCGCCTTCCAGCAGT

661 GGCCTTTCATATGTTTACCGGAGGATATTCAAGGGAGGGAAAACTATAGCAGCTGATAAT

721 GCACTGGATTATGCAGCAAATTTTTCACACATGCTTGGGTTTGATGATCCCAAAATGCTT

781 GAGTTGATGCGACTATATATAACAATCCACACTGATCATGAAGGTGGAAACGTCAGTGCT

841 CATACTGGACATCTGGTTGGAAGTGCTCTGTCAGACCCTTATCTTTCTTTTGCAGCTGCA

901 CTGAATGGTTTAGCTGGACCGTTGCACGGCCTGGCTAATCAGGAAGTGTTGTTGTGGATC

961 AAATCTGTAATAGGTGAGACTGGTAGTGACGTTACAACTGATCAACTCAAAGAGTATGTG

1021 TGGAAGACACTAAAAGGTGGAAAGGTTGTTCCTGGCTTTGGTCATGGAGTTCTACGTAAG

1081 ACCGATCCACGGTATACATGTCAGAGGGAGTTTGCTTTGAAGTACTTGCCTGAGGATCCA

1141 CTTTTCCAACTGGTCTCCAAGTTGTATGAAGTTGTGCCTCCTATCCTCACTGAGCTTGGC

1201 AAGGTCAAAAACCCATGGCCTAATGTTGATGCTCCACAGCGGAGTTCTACTGAACCACTTT

1261 GGATTATCTGAAGCTCGGTATTACACTGTTCTTTTCGGAGTTTCAAGGAGCATTGGAATA

1321 GGATCTCAGCTCATTTGGGACCGTGCTCTTGGCCTGCCGCTCGAAAGACCGAAGAGTGTC

1381 ACCATGGAGTGGCTGGAGAACCACTGCAAGAAGGTTGCTGCTTGA

(2) Information of SEQ ID NO.2

(i) Sequential features:

(A) Length: 474 amino acids

(B) type: amino acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: polypeptide

(iii) Sequence description: SEQ ID NO.2

1 MAFFRGLTAV SRLRSRVAQE ATTLGGVRWL QMQSASLDL KSQLQELIPE

51 QQDRLKKLKS EHGKVQLGNI TVDMVLGGMR GMTGMLWETS LLDPDEGIRF

101 RGLSIPERQK VLPTAVKDGE PLPEGLLWLL LTGKVPTKEQ VDALSKELAS

151 RSSVPGHVYE AIDALPVTAH PMTQFTTGVM ALQVESEFQK SPMTKGMSKS

201 KFWEPTYERL LKFDSSPSSS GLSYVYRRIF KGGKTIAADN ALDYAANFSH

251 MLGFDDPKML ELMRLYITIH TDHEGGNVSA HTGHLVGSAL SDPYLSFAAA

301 LNGLAGPLHG LANQEVLLWI KSVIGETGSD VTTDQLKEYV WKTLKGGKVV

351 PGFGHGVLRK TDPRYTCQRE FALKYLPEDP LFQLVSKLYE VVPPILTELG

401 KVKNPWPNVD AHSGVLLNHF GLSEARYYTV LFGVRSSIGI GSQLIWDRAL

451 GLPLERPKSV TMEWLENHCK KVAA

Claims (6)

1. An isolated DNA molecule is characterized in that it comprises: an open reading frame coding with rice citrate synthase activity, and its nucleotide sequence is SEQ ID NO.1. 2. DNA molecules as claimed in claim 1, is characterized in that, this sequence coding has the polypeptide of the aminoacid sequence shown in SEQ ID NO.2. 3. a kind of method that the nucleotide sequence that the coding as claimed in claim 1 has rice citrate synthase is transferred to tobacco to improve the amount of citric acid secretion is characterized in that concrete steps are as follows: (1) operably linking the nucleotide sequence encoding rice citrate synthase to the plant expression vector to form a vector containing SEQ ID NO.1; (2) Transform the expression vector in step (1) into Agrobacterium, co-cultivate the Agrobacterium containing the expression vector with tobacco leaves, and culture in dark for 2 days at 25±2°C; (3) Through antibiotic screening and PCR identification, transformed cells are obtained and transgenic plants are finally regenerated, and the citric acid secretion of the transgenic plants increases. 4. a method for detecting the copy number of rice citrate synthase in a sample is characterized in that it comprises hybridizing with the sequence described in claim 1 with the sample, and then whether the detection probe is combined; the sample is rice and Genomic DNA of transgenic tobacco, nucleotide sequence digested by restriction endonuclease. 5. The nucleic acid molecule for identifying transgenic tobacco is characterized in that it comprises 893-910 consecutive nucleotides and the reverse complement of 1405-1425 consecutive nucleotides in the DNA molecule shown in SEQ ID NO.1 sequence. 6. A method for detecting whether there is a rice citrate synthase nucleotide sequence in a sample, characterized in that it comprises carrying out a PCR reaction with the nucleotide sequence described in claim 5 and a sample, and then detecting whether the amplified The target fragment; the sample is transgenic tobacco group DNA.

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