CN107630033B

CN107630033B - Application of protein OsZFP213 in regulation and control of plant stress resistance

Info

Publication number: CN107630033B
Application number: CN201710982678.8A
Authority: CN
Inventors: 种康; 张泽勇; 马启斌; 徐云远
Original assignee: Institute of Botany of CAS
Current assignee: Institute of Botany of CAS
Priority date: 2017-10-20
Filing date: 2017-10-20
Publication date: 2020-01-31
Anticipated expiration: 2037-10-20
Also published as: CN107630033A

Abstract

The invention discloses application of protein OsZFP213 in regulating and controlling plant stress resistance, the protein OsZFP213 provided by the invention is a1) or a2) or a3), a1) amino acid sequence is protein shown in a sequence 2 in a sequence table, a2) fusion protein obtained by connecting labels at N end or/and C end of the protein shown in the sequence 2 in the sequence table, a3) protein related to plant stress resistance obtained by substituting and/or deleting and/or adding or a plurality of amino acid residues of the amino acid sequence shown in the sequence 2 in the sequence table, experiments prove that the OsZFP213 gene is overexpressed in No. 10 in rice varieties to obtain transgenic OsZFP213 rice, and the stress resistance of the transgenic OsZFP213 rice is enhanced compared with that of No. 10 in rice varieties, therefore, the protein OsP 213 has important theoretical significance and practical value in cultivating plants with enhanced stress resistance.

Description

Application of protein OsZFP213 in regulation and control of plant stress resistance

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of protein OsZFP213 in regulation and control of plant stress resistance.

Background

The salinization of soil becomes a serious problem influencing agricultural production, and the key problem and the important problem which need to be solved urgently in new variety cultivation are that the saline-alkali tolerance of crops is improved by utilizing a genetic engineering means, and the adaptability of the crops and the economic crops to adverse circumstances is improved. In recent years, people have made a lot of researches on the mechanism of plant responding to the adverse stress such as saline and alkaline from the aspects of physiology, biochemistry, metabolism, ecology, heredity, evolution and the like, accumulate abundant data, and particularly with the development of molecular biology, people can know the stress tolerance mechanism of plants to the salt stress at the molecular level of gene composition, expression regulation, signal conduction and the like, thereby opening up a new way for improving the stress resistance of plants by utilizing genetic engineering means. Due to the complexity of the stress resistance of plants, the traditional breeding method is very difficult to improve the stress resistance of the plants, with the development of molecular biology, a new approach for the stress resistance breeding of the plants is developed by a genetic engineering means, but the separation of high-efficiency stress resistance genes becomes a main factor for limiting the stress resistance genetic engineering of the plants.

Disclosure of Invention

The invention aims to solve the technical problem of how to improve the stress resistance of plants.

In order to solve the technical problems, the invention firstly provides the application of the protein OsZFP213 in regulating and controlling the stress resistance of plants.

In the above application, the protein OsZFP213 can be a1) or a2) or a3) or a 4):

a1) the amino acid sequence is protein shown as a sequence 2 in a sequence table;

a2) a fusion protein obtained by connecting labels to the N end or/and the C end of the protein shown in the sequence 2 in the sequence table;

a3) the protein which is obtained by substituting and/or deleting and/or adding or more amino acid residues to the amino acid sequence shown in the sequence 2 in the sequence table and is related to the plant stress resistance;

a4) the protein which has 80 percent or more than 80 percent of identity with the amino acid sequence limited by the sequence 2 in the sequence table, is derived from plants and is related to plant stress resistance.

Wherein, the sequence 2 in the sequence table is composed of 210 amino acid residues.

In order to facilitate the purification of the protein in a1), the amino terminal or the carboxyl terminal of the protein shown in the sequence 2 in the sequence table can be connected with a label shown in the table 1.

TABLE 1 sequence of tags

The protein of a3), wherein the or several amino acid residues are substituted and/or deleted and/or added by not more than 10 amino acid residues.

The protein of a3) above may be artificially synthesized, or may be obtained by synthesizing the coding gene and then performing biological expression.

The gene encoding the protein in a3) above can be obtained by deleting or several amino acid residues of the codon in the DNA sequence shown in sequence 1 in the sequence table, and/or performing or several base pairs missense mutation, and/or connecting the coding sequence of the tag shown in Table 1 above at the 5 'end and/or 3' end.

In the above application, the term "identity " is used to refer to sequence similarity with a natural amino acid sequence "identity " includes amino acid sequences having 80%, or 85% or more, or 90% or more, or 95% or more identity with the amino acid sequence shown in sequence No. 2 of the sequence Listing of the present invention identity can be evaluated visually or by computer software Using computer software, identity between two or more sequences can be expressed in percent (%) which can be used to evaluate identity between related sequences.

The application of the nucleic acid molecule for coding the protein OsZFP213 in regulating and controlling the stress resistance of plants also belongs to the protection scope of the invention.

In the above application, the nucleic acid molecule encoding the protein OsZFP213 can be a DNA molecule shown as b1) or b2) or b3) or b 4):

b1) the coding region is a DNA molecule shown as a sequence 1 in a sequence table;

b2) the nucleotide sequence is a DNA molecule shown as a sequence 1 in a sequence table;

b3) a DNA molecule which has 75% or more than 75% identity with the nucleotide sequence defined by b1) or (b2) and encodes the protein OsZFP 213;

b4) a DNA molecule which hybridizes with the nucleotide sequence defined in (b1) or (b2) under strict conditions and encodes the protein OsZFP 213.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

Wherein, the sequence 1 in the sequence table consists of 633 nucleotides, and the nucleotide of the sequence 1 in the sequence table encodes an amino acid sequence shown as a sequence 2 in the sequence table.

In the above applications, the term "identity " is used to refer to sequence similarity with a native nucleic acid sequence "identity " includes nucleotide sequences that have 75%, or 80% or more, or 85% or more, or 90% or more, or 95% or more identity to with the nucleotide sequence of the protein consisting of the amino acid sequence shown in sequence No. 2 of the sequence Listing of the present invention. identity can be evaluated visually or by computer software using computer software, identity between two or more sequences can be expressed as a percentage (%) which can be used to evaluate identity between related sequences.

In the above application, the regulation of the plant stress resistance may be an increase in the plant stress resistance.

In the above application, the stress resistance may be salt resistance.

In the application, the plant can be any of c1) to c5) as follows, c1) a dicotyledonous plant, c2) a monocotyledonous plant, c3) a gramineous plant, c4) rice and c5) No. 10 flowers in a rice variety.

In order to solve the technical problems, the invention also provides a method for cultivating the transgenic plant.

The method for cultivating the transgenic plant, which can be specifically the method for cultivating the transgenic plant, comprises the step of introducing a substance for improving the expression and/or activity of the protein OsZFP213 into a receptor plant to obtain a transgenic plant A, wherein the stress resistance of the transgenic plant A is improved compared with that of the receptor plant A.

In the method for breeding a transgenic plant, the "introducing a substance that increases the expression and/or activity of the protein OsZFP213 into a recipient plant" may be performed by introducing a nucleic acid molecule encoding the protein OsZFP213 into a recipient plant.

In the method for breeding transgenic plants, the nucleic acid molecule encoding the protein OsZFP213 can be a DNA molecule shown in b1) or b2) or b3) or b4) as follows:

In the above methods, the term "identity " is used to refer to sequence similarity with a native nucleic acid sequence "identity " includes nucleotide sequences that have 75%, or 80% or more, or 85% or more, or 90% or more, or 95% or more identity to with the nucleotide sequence of the protein consisting of the amino acid sequence shown in sequence No. 2 of the sequence Listing of the present invention. identity can be evaluated visually or with computer software using computer software, identity between two or more sequences can be expressed as a percentage (%) which can be used to evaluate identity between related sequences.

In the method for producing transgenic plants, the "introduction of the nucleic acid molecule encoding the protein OsZFP213 into the recipient plant" may be achieved by introducing a recombinant vector into the recipient plant, the recombinant vector may be a recombinant plasmid obtained by inserting the nucleic acid molecule encoding the protein OsZFP213 into an expression vector, and the recombinant vector may be specifically the overexpression vector pUN-ZFP213 (sense) mentioned in the examples.

The method for cultivating the transgenic plant, which can be specifically a method II for cultivating the transgenic plant, comprises the step of introducing a substance for inhibiting the expression and/or activity of the protein OsZFP213 into a receptor plant to obtain a transgenic plant B; the transgenic plant b has reduced stress resistance as compared to the recipient plant.

In the second method for producing a transgenic plant, the step of "introducing a substance inhibiting the expression and/or activity of the protein OsZFP213 into a recipient plant" may be carried out by introducing a recombinant vector into the recipient plant; the recombinant vector may be a recombinant plasmid obtained by inserting a reverse complement sequence of a nucleic acid molecule encoding the protein OsZFP213 into an expression vector. The recombinant vector may in particular be the overexpression vector pUN-ZFP213 (antisense) mentioned in the examples.

In order to solve the technical problems, the invention also provides a plant breeding method.

The plant breeding method comprises increasing the content and/or activity of OsZFP213 protein in plant, thereby improving stress resistance of plant.

In the plant breeding method , the "increasing the content and/or activity of the protein OsZFP213 in the plant" may be achieved by multiple copies, by modifying promoters, regulatory factors, transgenes, etc. by methods well known in the art, such that the content and/or activity of the protein OsZFP213 in the plant is increased.

The plant breeding method provided by the invention can be specifically a plant breeding method II, and comprises the following steps: reducing the content and/or activity of the protein OsZFP213 in the plant, thereby reducing the stress resistance of the plant.

In the second plant breeding method, the "reducing the content and/or activity of the protein OsZFP213 in the plant" can be achieved by methods known in the art, such as RNA interference, homologous recombination, gene site-directed editing, and the like, so as to achieve the purpose of reducing the content and/or activity of the protein OsZFP 213.

In any of the methods above, the stress resistance can be salt resistance.

In the method of any , the recipient plant may be any plants selected from c1) to c5), c1) a dicotyledonous plant, c2) a monocotyledonous plant, c3) a gramineous plant, c4) rice, and c5) a cultivar medium-flowering-10.

Experiments prove that the OsZFP213 gene is overexpressed in No. 10 flowers in rice varieties to obtain OsZFP213 gene-transferred rice; compared with the rice variety No. 10, the OsZFP213 transgenic rice has enhanced stress resistance. Therefore, the protein OsZFP213 has important theoretical significance and practical value in cultivating plants with enhanced stress resistance.

Drawings

FIG. 1 shows the result of agarose gel electrophoresis of the PCR amplification product A in step three of example 1.

FIG. 2 is a schematic diagram of the structure of the overexpression vector pUN-ZFP213 (sense) and the expression vector pUN-ZFP213 (antisense).

FIG. 3 shows the results of the third step of example 2.

FIG. 4 shows the results of the experiment in step four of example 2.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Zhonghua No. 10 (plum blossom. cultivar of rice blossom-Zhonghua No. 10, agricultural science and technology communications, p.26 of No. 1 of 1998) is a rice variety, which was publicly available from plant research institute of Chinese academy of sciences to repeat the experiment, Zhonghua No. 10 is hereinafter referred to as ZH 10.

The DNA gel recovery kit is a product of AxyPrep company. The vector pBI121 is a product of Biotechnology, Inc. of Byeldi, Beijing, and the catalog number is MP-091. The vector pUC19 was a product of Biotechnology Inc., Baitach, Beijing, under the catalog number DP 7801. The RT-PCR kit is a product of Promega corporation. The Trizol reagent is a product of Invitrogen corporation. Vector pCAMBIA1301 is Biovector Co., LTD, catalog number Biovec-11. The EasyJect Plus electro-stimulator is a product of EquiBio Inc. of UK. LA Taq, 2 XGC buffer and dNTPs are all products of TaKaRa.

The specification of the centrifuge tube is 1.5 mL.

MS liquid culture medium: reacting NH₄NO₃1650mg、KNO₃1900mg、KH₂PO₄170mg、MgSO₄·7H₂O 370mg、CaCl₂·2H₂O 440mg、FeSO₄·7H₂O 27.8mg、Na₂EDTA 37.3mg、MnSO₄·4H₂O 22.3mg、ZnSO₄·7H₂O 8.6mg、H₃BO₃6.2mg、KI 0.83mg、Na₂MoO₄·2H₂O 0.25mg、CuSO₄·5H₂O 0.025mg、CoCl₂·6H₂O0.025 mg, inositol 100.00mg, VB₁0.5mg、VB₆0.5mg, nicotinic acid 0.5mg, glycine 2.0mg and sucrose 30g were dissolved in 1L of distilled water, and pH was adjusted to 5.8.

MS solid culture medium: agar was added to the MS liquid medium so that the concentration of agar in the medium became 8 g/L.

N6D2 medium: MS solid medium containing 300mg/L hydrolyzed casein, 500mg/L proline, 500mg/L glutamine, 30g/L sucrose and 2 mg/L2, 4-D.

N6D2S1 medium: N6D2 medium containing 25mg/L hygromycin and 600mg/L cephamycin.

N6D2S2 medium: N6D2 medium containing 50mg/L hygromycin and 300mg/L cephamycin.

Differentiation medium (1): N6D2 culture medium containing 300mg/L hydrolyzed casein, 50mg/L hygromycin, 1 mg/L6-BA, 0.5mg/L KT, 0.2mg/L ZT, 0.25mg/L NAA, 30g/L sucrose and 30g/L sorbitol.

Differentiation medium (2): N6D2 culture medium containing 300mg/L hydrolyzed casein, 50mg/L hygromycin, 1 mg/L6-BA, 0.5mg/L KT, 0.2mg/L ZT, 0.5mg/L NAA, 30g/L sucrose and 20g/L sorbitol.

Rooting and seedling strengthening culture medium: 1/2MS solid medium containing 1mg/L paclobutrazol and 0.5mg/L NAA.

The Mucuna B culture medium is described in the following documents: ma JF, Goto S, Tamain K, Ichi M.role of roads and laterals in silicon uptake by rice plant physiology.2001; 127: 1773 and 1780) in the formulation shown in Table 2, at a pH of 5.8.

TABLE 2

Example 1 obtaining of OsZFP213 Gene

Synthesis of primers

Artificially synthesizing a primer F1: 5' -CGGGATCCATGGAGGCTCCCCCTTCTCT-3' (recognition site for restriction enzyme BamHI is underlined) and primer R1: 5' -GGGGTACCGAGCTTCAGGTTGAGATCAACCTGC-3' (the recognition site for the restriction enzyme KpnI is underlined).

Artificially synthesizing a primer F2: 5' -CGGGATCCGAGCTTCAGGTTGAGATCAACCTGC-3' (recognition site for restriction enzyme BamHI is underlined) and primer R2: 5' -GGGGTACCATGGAGGCTCCCCCTTCTCT-3' (the recognition site for the restriction enzyme KpnI is underlined).

Secondly, obtaining a template

Extracting total RNA of seedlings with a medium flower number 10 trilobate stage, and then reversely transcribing the total RNA to th chain cDNA, the specific steps are as follows:

1. 100mg of seedling of the middle flower in the No. 10 trilobate stage is taken and ground by liquid nitrogen to obtain freeze-dried powder.

2. After the step 1 is completed, transferring the freeze-dried powder into a centrifuge tube filled with 1mL of Trizol reagent, fully and uniformly mixing, and standing for 5min at 25 ℃.

3. After the step 2 is finished, taking the centrifuge tube, adding 0.2mL of fresh chloroform, violently shaking for 15s, and incubating for 2-3min at 25 ℃; then, the mixture was centrifuged at 12000rpm for 15min at 4 ℃.

4. After completion of step 3, the supernatant (about 0.5mL) was transferred to a centrifuge tube, 0.5mL isopropanol was added, and the mixture was allowed to stand at 25 ℃ for 10 min; then, the mixture was centrifuged at 12000rpm for 15min at 4 ℃.

5. After the step 4 is finished, discarding the supernatant, washing the precipitate for 2 times by using 1mL of 75% (v/v) ethanol aqueous solution, and blowing the precipitate to be semi-dry by using an ultra-clean bench; then 50mL DEPC-ddH was used₂O heavy suspension, 60 ℃ water bath for 10min, and obtaining RNA solution.

6. After completing the step 5, performing reverse transcription according to the operation steps of the RT-PCR kit instruction to obtain chain cDNA:

(1) the RNA solution (containing 2. mu.g of gRNA) was added with 1.0. mu.g of Oligo dT primer, followed by DEPC-ddH₂Supplementing O to 15 μ L, mixing, denaturing at 70 deg.C for 5min, and ice-cooling for 5 min.

(2) To the system for completion of step (1), 25. mu.L of a reverse transcription Mixture (composed of 5. mu. L M-MLV 5 × Reactionbuffer, 6. mu.L of dNTP mix (concentrations of dATP, dTTP, dGTP and dCTP are each 2.5mM), 1. mu. L M-MLVReverse Transcriptase, 0.5. mu.L of RNase Inhibitor and 12.5. mu.L of DEPC-ddH were added₂O), mixing, water bath at 42 deg.C for 1h (for reverse transcription), and water bath at 75 deg.C for 10min (for reverse transcriptase inactivation) to obtain th chain cDNA.

Oligo dT primer, M-MLV 5 × Reaction Buffer, dNTP mix, M-MLV reverse transcription and RNase Inhibitor are all components in RT-PCR kit.

Third, PCR amplification reaction

1. And (3) performing PCR amplification by using the th chain cDNA obtained in the step two as a template and adopting a primer pair A (consisting of a primer F1 and a primer R1) to obtain a PCR amplification product A.

Reaction system (20 μ L): 0.2. mu.L of LA Taq (concentration 5U/. mu.L), 10. mu.L of 2 XGC buffer, 1.8. mu.L of dNTPs (concentrations of dATP, dTTP, dGTP and dCTP are all 2.5mM), 0.5. mu.L ofPrimer F1 (10. mu.M concentration), 0.5. mu.L primer R1 (10. mu.M concentration) and 7. mu.L ddH₂And (C) O.

Reaction conditions are as follows: pre-denaturation at 94 ℃ for 30 s; denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 40s, and 35 cycles; extension at 72 ℃ for 10 min.

2. And (3) performing PCR amplification by using the th chain cDNA obtained in the step (II) as a template and adopting a primer pair B (consisting of a primer F2 and a primer R2) to obtain a PCR amplification product B.

Reaction system (20 μ L): mu.L LA Taq (5U/. mu.L concentration), 10. mu.L 2 XGC buffer, 1.8. mu.L dNTPs, 0.5. mu.L primer F2 (10. mu.M concentration), 0.5. mu.L primer R2 (10. mu.M concentration) and 7. mu.L ddH₂And (C) O.

The reaction conditions are the same as those of step 1.

Subjecting the PCR amplification product A or the PCR amplification product B to 0.8% (m/m) agarose gel electrophoresis. Part of the experimental results are shown in FIG. 1(Marker is DNA Marker, and 1 is PCR amplification product A). The result shows that the sizes of the PCR amplification product A and the PCR amplification product B are 649 bp.

And recovering the PCR amplification product A or the PCR amplification product B by adopting a DNA gel recovery kit, and then sequencing. The sequencing result shows that the PCR amplification product A and the PCR amplification product B both contain nucleotide sequences shown in a sequence 1 in a sequence table. The gene shown in the sequence 1 in the sequence table is named as OsZFP213 gene, the protein coded by the gene is named as OsZFP213 protein or protein OsZFP213, and the amino acid sequence is shown as the sequence 2 in the sequence table.

Example 2 application of OsZFP213 protein in regulation and control of rice salt resistance

construction of recombinant plasmid

1. Construction of recombinant plasmid pUN1301

(1) Extracting the genome DNA of corn seedling and using it as template, using 5' -GGAAGCTTCTGCAGTGCAGCGTGACCCGG-3 '(recognition sites for the restriction enzyme HindIII are underlined) and 5' -CGGGATCCAAGTAACACCAAACAACAGGG-3' (underlined is a recognition site of a restriction enzyme BamHI) as a primer, and PCR amplification products were obtained.

(2) After completion of step (1), the PCR amplification product was digested with restriction enzymes HindIII and BamHI, and a DNA fragment A of about 2kb was recovered by using a DNA gel recovery kit.

Sequencing the DNA fragment A. The sequencing result shows that the DNA fragment A contains a nucleotide sequence shown as a sequence 3 in a sequence table. The nucleotide sequence shown in the sequence 3 in the sequence table is a maize ubiquitin promoter (Ubipro).

(3) The vector pBI121 is cut by restriction enzymes SacI and EcoRI, and a DNA gel recovery kit is adopted to recover a DNA fragment B (namely Noster poly A) with about 260 bp.

(4) The vector pUC19 was digested with restriction enzymes SacI and EcoRI, and the vector backbone A of about 2686bp was recovered using a DNA gel recovery kit.

(5) And connecting the DNA fragment B with the vector framework A to obtain a recombinant plasmid pUC 19-Noster.

(6) The recombinant plasmid pUC19-Noster was digested with restriction enzymes HindIII and BamHI, and vector backbone B of about 2700bp was recovered using a DNA gel recovery kit.

(7) And connecting the DNA fragment A with the vector framework B to obtain a recombinant plasmid pUN 19.

(8) The recombinant plasmid pUN19 was digested with restriction enzymes EcoRI and HindIII, and a DNA fragment C of about 2.3kb was recovered using a DNA gel recovery kit.

(9) The vector pCAMBIA1301 was digested with restriction enzymes EcoRI and HindIII, and the vector backbone C of about 11.8kb was recovered using a DNA gel recovery kit.

(10) And connecting the DNA fragment C with a vector skeleton C to obtain a recombinant plasmid pUN 1301.

2. Construction of overexpression vector pUN-ZFP213 (sense)

A schematic diagram of the structure of the over-expression vector pUN-ZFP213 (sense) is shown in FIG. 2A (Ubi is the maize ubiquitin promoter and NOS is Noster poly A).

(1) The recombinant plasmid pUN1301 was digested with restriction enzymes KpnI and BamHI, and the vector backbone 1 of about 14kb was recovered using a DNA gel recovery kit.

(2) The PCR amplification product A recovered in the third step of example 1 was digested with restriction enzymes KpnI and BamHI, and a DNA fragment 1 of about 650kb was recovered by using a DNA gel recovery kit.

(3) The vector backbone 1 and the DNA fragment 1 were ligated to obtain the over-expression vector pUN-ZFP213 (sense).

The overexpression vector pUN-ZFP213 (sense) was sequenced. Based on the sequencing results, the overexpression vector pUN-ZFP213 (sense) was structurally described as follows: the small fragment between the recognition sequences of the restriction enzymes KpnI and BamHI of the recombinant plasmid pUN1301 was replaced with a DNA molecule shown as sequence 1 in the sequence listing. The overexpression vector pUN-ZFP213 (sense) expresses OsZFP213 protein shown in a sequence 2 in a sequence table.

The over-expression vector pUN-ZFP213 (sense) has expression cassettes A, the nucleotide sequence of the expression cassettes A is shown as a sequence 4 in a sequence table, wherein, the 1 st to 1987 th sites of the sequence 4 in the sequence table from the 5' end are maize ubiquitin promoters, the 1994 th to 2626 th sites are coding genes of OsZFP213 protein, and the 2639 th to 2909 th sites are terminator sequences of Nos genes.

3. Construction of expression vector pUN-ZFP213 (antisense)

The expression vector pUN-ZFP213 (antisense) has a structure as shown in B in FIG. 2 (Ubi is the maize ubiquitin promoter and NOS is Noster poly A).

(2) The PCR amplification product B recovered in the third step of example 1 was digested with restriction enzymes KpnI and BamHI, and a DNA fragment 2 of about 650kb was recovered by using a DNA gel recovery kit.

(3) The vector backbone 1 and the DNA fragment 2 were ligated to obtain the expression vector pUN-ZFP213 (antisense).

The expression vector pUN-ZFP213 (antisense) was sequenced. Based on the sequencing results, the expression vector pUN-ZFP213 (antisense) was structurally described as follows: the small fragment between the recognition sequences of the restriction enzymes KpnI and BamHI of the recombinant plasmid pUN1301 was replaced with the reverse complement of the DNA molecule shown in sequence 1 of the sequence listing.

Two, T₀Generation of transgenic OsZFP213 Rice

1、T₀Generation of transgenic OsZFP213 Rice (sense)

(1) And (3) transforming the overexpression vector pUN-ZFP213 (sense) into the agrobacterium tumefaciens EHA105 by adopting an EasyJect Plus electrical stimulator to obtain a recombinant agrobacterium A, and naming the recombinant agrobacterium A as EHA105/pUN-OsZFP213 (sense).

(2) After the step (1) is completed, EHA105/pUN-OsZFP213 (sense) is introduced into the calli of Zhonghua No. 10 by adopting an agrobacterium-mediated method, the calli are washed for 4-5 times by using sterile water containing 300mg/L of cefamycin, the calli are blotted by sterile filter paper and transferred to an N6D2S1 culture medium, and the 1 generation is screened (2 weeks).

(3) After completion of step (2), the calli were transferred to N6D2S2 medium and screened for 2 passages, 2 weeks per passage.

(4) After the step (3) is finished, the vigorous resistant callus is firstly transferred to a differentiation culture medium (1) and cultured for 7d in a differentiation culture box (12 hours photoperiod, illumination intensity is 8000 lux; day 28 ℃, night 25 ℃); then transferred to a differentiation medium (2) and cultured in a differentiation incubator (12 hours photoperiod, illumination intensity of 8000 lux; day 28 ℃, night 25 ℃) until a regenerated seedling grows out.

(5) After the step (4) is finished, the regenerated seedlings take roots and are strong on a rooting and strong seedling culture medium; opening the sealing film of the container when the seedlings grow to about 10c m, hardening the seedlings for 2-3 days, and then transferring the seedlings into a phytotron for cultivation.

Through the steps, 60T plants in 10 strains are obtained₀Transgenic OsZFP213 rice (sense).

2、T₀Generation of transgenic OsZFP213 Rice (antisense)

According to the above step 1, "EHA 105/pUN-OsZFP213 (sense)" was replaced with EHA105/pUN-OsZFP213 (antisense), and the other steps were not changed, to obtain 100T strains of 10 strains in total₀Transgenic OsZFP213 rice (antisense).

The EHA105/pUN-OsZFP213 (antisense) was prepared as follows: and (3) transforming the expression vector pUN-ZFP213 (antisense) into the agrobacterium tumefaciens EHA105 by adopting an EasyJect Plus electrical stimulator to obtain a recombinant agrobacterium B, and naming the recombinant agrobacterium B as EHA105/pUN-OsZFP213 (antisense).

3、T₀Obtaining of rice with simulated empty carrier

Replacing EHA105/pUN-OsZFP213 (sense) "with EHA105/pUN1301 in the above step 1, and obtaining T in the same manner as the other steps₀The generation simulates rice with an empty carrier.

The preparation method of EHA105/pUN1301 is as follows: the recombinant plasmid pUN1301 is transformed into agrobacterium tumefaciens EHA105 by adopting an EasyJect Plus electrical stimulator to obtain recombinant agrobacterium tumefaciens C, and the recombinant agrobacterium tumefaciens C is named as EHA105/pUN 1301.

III, T₀Identification of transgenic OsZFP213 rice

1. GUS histochemical staining

GUS staining solution: pH7.0 containing 0.1% (v/v) Triton X-100, 10mM EDTA, 0.5mM potassium ferrocyanide, 0.5mM potassium ferricyanide and 1mg/mL X-Gluc, 100mM Na₃PO₄And (4) a buffer solution.

A、T₀GUS histochemical staining for transgenic OsZFP213 rice (sense)

(1) Respectively combining 60 strains of the 10 strains obtained in the step two in total₀Placing root segments (about 2-3mm in length) of the pseudo-trans OsZFP213 rice (sense) in GUS staining solution, and exhausting for several minutes; then incubated at 37 ℃ overnight, decolorized with 70% (v/v) aqueous ethanol, and judged as follows: if the root segment is blue, then the corresponding T₀The pseudo-transgenic OsZFP213 rice (sense) is T₀Transgenic OsZFP213 rice (sense).

50T of 6 strains are obtained through GUS histochemical staining identification₀Transgenic OsZFP213 rice (sense).

(2) Identifying step (1) as T₀Transferring OsZFP213 rice (positive sense) to greenhouse for cultivation, and harvesting according to different strains to obtain T₁Transgenic OsZFP213 rice (sense) seeds. Will T₁Seeds of transgenic OsZFP213 rice (sense) are bred to obtain T₂Transgenic OsZFP213 rice (sense) with homozygous generations.

Get T₀Transgenic OsZFP213 rice (sense)) T of strains 3 (abbreviated to OE3) and 7 (abbreviated to OE7) of₂The generation homozygous seeds are used as experimental materials for subsequent experiments.

B、T₀GUS histochemical staining for transgenic OsZFP213 rice (antisense)

(1) Respectively combining the 10 strains obtained in the step two 1 to obtain 100T plants₀Placing root segments (about 2-3mm in length) of OsZFP213 rice (antisense) in GUS staining solution, and pumping for several minutes; then incubated at 37 ℃ overnight, decolorized with 70% (v/v) aqueous ethanol, and judged as follows: if the root segment is blue, then the corresponding T₀The generation-simulated OsZFP213 rice (antisense) is T₀Transgenic OsZFP213 rice (antisense).

Through GUS histochemical staining identification, 80T plants of 8 strains are obtained₀Transgenic OsZFP213 rice (antisense).

(2) Identifying step (1) as T₀Transferring OsZFP213 rice (antisense) to greenhouse for cultivation, and harvesting according to different strains to obtain T₁Transgenic OsZFP213 rice (antisense) seeds. Will T₁Seeds of trans-OsZFP 213 rice (antisense) are bred to obtain T₂Transgenic OsZFP213 rice (antisense) with homozygous generations.

Get T₀T of line 1 (abbreviated AS AS1) and line 3 (abbreviated AS AS3) of transgenic OsZFP213 rice (antisense)₂The generation homozygous seeds are used as experimental materials for subsequent experiments.

C、T₀GUS histochemical staining for rice with simulated empty vector

(1) Respectively mixing T obtained in step two₀Placing root segments (about 2-3mm in length) of rice with simulated empty vector in GUS staining solution, and exhausting for several minutes; then incubated at 37 ℃ overnight, decolorized with 70% (v/v) aqueous ethanol, and judged as follows: if the root segment is blue, then the corresponding T₀The rice with the simulated empty carrier is T₀Transferring the rice with empty carrier.

(2) Identifying step (1) as T₀Transferring the rice with the empty carrier to a greenhouse for cultivation, and harvesting according to different strains to obtain T₁Transferring the seeds of the rice with the empty carrier. Will T₁The seeds of the rice with the empty carrier are obtained by seed reproductionTo T₂The generation pure rice is transferred to empty carrier rice.

Get T₂Seeds of the generation homozygous empty carrier rice are used as experimental materials for subsequent experiments.

2. Semi-quantitative PCR

A、T₂Semi-quantitative PCR of generation-homozygous OsZFP213 rice (sense)

The rice seedling to be detected is OE3 seedling (T of OE3)₂Germination of generation homozygous seed), OE7 seedling (T of OE7)₂Germination of generation homozygous seeds), and empty vector-transferred rice seedlings (T)₂Seed germination of generation homozygous empty carrier rice) or seedling No. 10 (seed germination of No. 10 seed).

1. Extracting mRNA of the rice seedling to be detected, and then carrying out reverse transcription to obtain cDNA.

2. And detecting the relative expression quantity of the OsZFP213 gene in each rice seedling to be detected by adopting a semi-quantitative PCR method (taking the TUBULIN gene as an internal reference gene).

Primers for detecting the OsZFP213 gene are 5'-ATGGAGGCTCCCCCTTCTCT-3' and 5'-GAGCTTCAGGTTGAGATCAACCTGC-3'. Primers for detecting the TUBULIN gene were 5'-TCAGATGCCCAGTGACAGGA-3' and 5'-TTGGTGATCTCGGCAACAGA-3'.

Reaction program for detecting OsZFP213 gene: pre-denaturation at 94 ℃ for 30 s; denaturation at 94 ℃ for 10s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 40s, for 27 cycles; extension at 72 ℃ for 10 min.

Reaction procedure for detection of TUBULIN gene: pre-denaturation at 94 ℃ for 30 s; denaturation at 94 ℃ for 10s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 40s for 25 cycles; extension at 72 ℃ for 10 min.

The results are shown in A of FIG. 3 (WT is middle flower No. 10). The experimental result shows that compared with seedlings of Zhonghua No. 10, the relative expression amounts of OsZFP213 genes in seedlings of OE3 and OE7 are obviously increased, and the relative expression amounts of OsZFP213 genes in empty vector rice seedlings are not obviously different. Thus, the OsZFP213 gene has been successfully expressed at the transcriptional level in seedlings of OE3 and in seedlings of OE 7.

B、T₂Semi-quantitative PCR (polymerase chain reaction) for generation homozygous transgenic OsZFP213 rice (antisense)

The rice seedling to be detected is AS1 seedling(T of AS1₂Germination of generation homozygous seed), AS3 seedling (T of AS3)₂Germination of generation homozygous seeds), and empty vector-transferred rice seedlings (T)₂Seed germination of generation homozygous empty carrier rice) or seedling No. 10 (seed germination of No. 10 seed).

The reaction programs for detecting the OsZFP213 gene and the TUBULIN gene are as follows: pre-denaturation at 94 ℃ for 30 s; denaturation at 94 ℃ for 10s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 40s for 30 cycles; extension at 72 ℃ for 10 min.

The results are shown in B of FIG. 3 (WT is middle flower No. 10). The experimental result shows that compared with the seedling of the medium flower No. 10, the relative expression quantity of the OsZFP213 gene in the AS1 seedling and the AS3 seedling is obviously reduced, and the relative expression quantity of the OsZFP213 gene in the empty vector rice seedling has no obvious difference. Thus, in seedlings AS1 and AS3, the OsZFP213 gene had been successfully expressed at the transcriptional level and decreased the expression of endogenous OsZFP213 gene transcripts.

Four, T₂Salt resistance identification of generation homozygous OsZFP213 rice

The parameters of light-dark alternate culture are as follows: the illumination period is 16h/8h, the temperature is 30 ℃, and the illumination intensity is 10000 mu mol/m²/s。

The rice seed to be detected is T of AS1₂T of generation homozygous seed, AS3₂Generation homozygous seed, T of OE3₂Generation homozygous seed, T of OE7₂Generation homozygous seed, T₂The generation homozygous empty carrier rice seeds or the middle flower No. 10 seeds.

The experiment was repeated 4 times to obtain an average, and the procedure for each repetition was as follows:

1. taking 24 rice seeds to be detected, subpackaging the seeds in kraft paper bags, and soaking the seeds for 48 hours at the temperature of 28-30 ℃.

2. After the step 1 is finished, accelerating germination for 24 hours at 28-30 ℃ (the seeds need to be kept wet in the accelerating germination process), and obtaining the accelerated germination seeds.

3. After step 2, a 96-well plate is taken, a part of the lower edge of each well is cut off, and then 1 seed (with embryo upward and radicle downward) which is accelerated to sprout is put into each well.

4. After the step 3 is completed, the 96-well plate (with the seeds which are accelerated to sprout) is placed on a plastic box containing the Mucuna B culture solution, the seeds which are accelerated to sprout are soaked in the Mucuna B culture solution, and light and dark alternate culture is carried out for 3 weeks, so that the rice seedlings which grow to the stage of three leaves are obtained. During the light-dark alternate culture period, the culture solution of Mucun B was changed every 7 days.

5. After completion of step 4, the 96-well plate (on which the rice seedlings grown to the trefoil stage were grown) was placed on a plastic box containing the culture solution of Mucun B containing 200mM NaCl and roots were completely immersed in the culture solution of Mucun B containing 200mM NaCl, and subjected to high salt stress for 11d under light-dark alternate culture (during the high salt stress period, the culture solution of Mucun B containing 200mM NaCl was changed times every 2 d).

6. After completion of step 5, the 96-well plate (with the rice seedlings thereon) was placed on a plastic box containing the Mucun B culture solution and the roots were completely immersed in the Mucun B culture solution and recovered for 14d under light-dark alternate culture.

And observing the growth state of the rice seedlings and counting the survival rate. Survival rate is the number of surviving rice seedlings/24 × 100%.

The growth state of rice seedlings is shown in A in FIG. 4. The survival statistics are shown in fig. 4B.

The results show that the growth states of the rice seedlings have no obvious difference before high salt stress; after high salt stress, AS1 seedlings (T of AS1)₂Germination of generation homozygous seed), AS3 seedling (T of AS3)₂Germination of generation homozygous seeds), and empty vector-transferred rice seedlings (T)₂Seed germination of generation homozygous empty vector rice) and seedling No. 10 of Zhonghua (seed germination of Zhonghua No. 10) with the leaves mostly whitened, and seedling OE3 (T of OE3)₂Germination of generation homozygous seeds) and OE7 seedlings (T of OE7)₂Germination of generation homozygous seeds) has mild leaf symptoms, and most of the leaves are green. After high salt stress, the survival rates of the OE3 seedlings and OE7 seedlings were significantly enhanced (51% and 55%, respectively) compared to the survival rate of medium flower No. 10 seedlings (29%), the survival rates of AS1 seedlings and AS3 seedlings were reduced (26% and 22%, respectively), and the survival rates of empty vector-transferred rice seedlings were not significantly different.

The above results show that T₂The salt resistance of the generation homozygous trans-OsZFP 213 rice (sense) is greatly improved.

<110> institute of plant of Chinese academy of sciences

Application of <120> protein OsZFP213 in regulation and control of plant stress resistance

<160>4

<170>PatentIn version 3.5

<210>1

<211>633

<212>DNA

<213> Rice (Oryza sativa L.)

<400>1

atggaggctc ccccttctct ttccatcgtc gacgaagacg gcttcgtcat cgacctctcg 60

ctgacgctgg gcctgacatc gcctccgccc tctccaggcg gcgcgtctcc ttccattcct 120

ccgggaagag gcggcggcgg cggcacgagt ggtggtgaca acaacagagg cagcagaggt 180

ggtggcaatg gtggtggtgg tggtggtgtg aggctcttcc cttgcctctt ctgcaacaag 240

aagtttctca agtcgcaggc gctaggggga caccagaatg cacacaagaa ggagaggagc 300

gtcgggtgga acacccacct ctacctcccg gccggcgtcg ccgccgccac gacgacgacg 360

acgacgacga cggcgatggc cgtccctgac atggtcggca tgcccaccca ccagatgtcc 420

tccatggcgt tgcactcctg ccggcctcac cagggctccc atgtcaccgc cgccgacatc 480

gccacgctgg ctgcgccgcc acattacacc gtcgaccatg gcgttgccgg catcgccagc 540

ggcggtggtg acagctcggt ggggtggcgc caaaggcaaa gggaggccgg cggcgagaag 600

cagaggcagg ttgatctcaa cctgaagctc tag 633

<210>2

<211>210

<212>PRT

<213> Rice (Oryza sativa L.)

<400>2

Met Glu Ala Pro Pro Ser Leu Ser Ile Val Asp Glu Asp Gly Phe Val

1 5 10 15

Ile Asp Leu Ser Leu Thr Leu Gly Leu Thr Ser Pro Pro Pro Ser Pro

20 25 30

Gly Gly Ala Ser Pro Ser Ile Pro Pro Gly Arg Gly Gly Gly Gly Gly

35 40 45

Thr Ser Gly Gly Asp Asn Asn Arg Gly Ser Arg Gly Gly Gly Asn Gly

50 55 60

Gly Gly Gly Gly Gly Val Arg Leu Phe Pro Cys Leu Phe Cys Asn Lys

65 70 75 80

Lys Phe Leu Lys Ser Gln Ala Leu Gly Gly His Gln Asn Ala His Lys

85 90 95

Lys Glu Arg Ser Val Gly Trp Asn Thr His Leu Tyr Leu Pro Ala Gly

100 105 110

Val Ala Ala Ala Thr Thr Thr Thr Thr Thr Thr Thr Ala Met Ala Val

115 120 125

Pro Asp Met Val Gly Met Pro Thr His Gln Met Ser Ser Met Ala Leu

130 135 140

His Ser Cys Arg Pro His Gln Gly Ser His Val Thr Ala Ala Asp Ile

145 150 155 160

Ala Thr Leu Ala Ala Pro Pro His Tyr Thr Val Asp His Gly Val Ala

165 170 175

Gly Ile Ala Ser Gly Gly Gly Asp Ser Ser Val Gly Trp Arg Gln Arg

180 185 190

Gln Arg Glu Ala Gly Gly Glu Lys Gln Arg Gln Val Asp Leu Asn Leu

195 200 205

Lys Leu

　　210

<210>3

<211>1987

<212>DNA

<213> corn (Zea mays L.)

<400>3

ctgcagtgca gcgtgacccg gtcgtgcccc tctctagaga taatgagcat tgcatgtcta 60

agttataaaa aattaccaca tatttttttt gtcacacttg tttgaagtgc agtttatcta 120

tctttataca tatatttaaa ctttactcta cgaataatat aatctatagt actacaataa 180

tatcagtgtt ttagagaatc atataaatga acagttagac atggtctaaa ggacaattga 240

gtattttgac aacaggactc tacagtttta tctttttagt gtgcatgtgt tctccttttt 300

ttttgcaaat agcttcacct atataatact tcatccattt tattagtaca tccatttagg 360

gtttagggtt aatggttttt atagactaat ttttttagta catctatttt attctatttt 420

agcctctaaa ttaagaaaac taaaactcta ttttagtttt tttatttaat aatttagata 480

taaaatagaa taaaataaag tgactaaaaa ttaaacaaat accctttaag aaattaaaaa 540

aactaaggaa acatttttct tgtttcgagt agataatgcc agcctgttaa acgccgtcga 600

cgagtctaac ggacaccaac cagcgaacca gcagcgtcgc gtcgggccaa gcgaagcaga 660

cggcacggca tctctgtcgc tgcctctgga cccctctcga gagttccgct ccaccgttgg 720

acttgctccg ctgtcggcat ccagaaattg cgtggcggag cggcagacgt gagccggcac 780

ggcaggcggc ctcctcctcc tctcacggca ccggcagcta cgggggattc ctttcccacc 840

gctccttcgc tttcccttcc tcgcccgccg taataaatag acaccccctc cacaccctct 900

ttccccaacc tcgtgttgtt cggagcgcac acacacacaa ccagatctcc cccaaatcca 960

cccgtcggca cctccgcttc aaggtacgcc gctcgtcctc cccccccccc cctctctacc 1020

ttctctagat cggcgttccg gtccatggtt agggcccggt agttctactt ctgttcatgt 1080

ttgtgttaga tccgtgtttg tgttagatcc gtgctgctag cgttcgtaca cggatgcgac 1140

ctgtacgtca gacacgttct gattgctaac ttgccagtgt ttctctttgg ggaatcctgg 1200

gatggctcta gccgttccgc agacgggatc gatttcatga ttttttttgt ttcgttgcat 1260

agggtttggt ttgccctttt cctttatttc aatatatgcc gtgcacttgt ttgtcgggtc 1320

atcttttcat gctttttttt gtcttggttg tgatgatgtg gtctggttgg gcggtcgttc 1380

tagatcggag tagaattctg tttcaaacta cctggtggat ttattaattt tggatctgta 1440

tgtgtgtgcc atacatattc atagttacga attgaagatg atggatggaa atatcgatct 1500

aggataggta tacatgttga tgcgggtttt actgatgcat atacagagat gctttttgtt 1560

cgcttggttg tgatgatgtg gtgtggttgg gcggtcgttc attcgttcta gatcggagta 1620

gaatactgtt tcaaactacc tggtgtattt attaattttg gaactgtatg tgtgtgtcat 1680

acatcttcat agttacgagt ttaagatgga tggaaatatc gatctaggat aggtatacat 1740

gttgatgtgg gttttactga tgcatataca tgatggcata tgcagcatct attcatatgc 1800

tctaaccttg agtacctatc tattataata aacaagtatg ttttataatt attttgatct 1860

tgatatactt ggatgatggc atatgcagca gctatatgtg gattttttta gccctgcctt 1920

catacgctat ttatttgctt ggtactgttt cttttgtcga tgctcaccct gttgtttggt 1980

gttactt 1987

<210>4

<211>2909

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>4

ctgcagtgca gcgtgacccg gtcgtgcccc tctctagaga taatgagcat tgcatgtcta 60

agttataaaa aattaccaca tatttttttt gtcacacttg tttgaagtgc agtttatcta 120

tctttataca tatatttaaa ctttactcta cgaataatat aatctatagt actacaataa 180

tatcagtgtt ttagagaatc atataaatga acagttagac atggtctaaa ggacaattga 240

gtattttgac aacaggactc tacagtttta tctttttagt gtgcatgtgt tctccttttt 300

ttttgcaaat agcttcacct atataatact tcatccattt tattagtaca tccatttagg 360

gtttagggtt aatggttttt atagactaat ttttttagta catctatttt attctatttt 420

agcctctaaa ttaagaaaac taaaactcta ttttagtttt tttatttaat aatttagata 480

taaaatagaa taaaataaag tgactaaaaa ttaaacaaat accctttaag aaattaaaaa 540

aactaaggaa acatttttct tgtttcgagt agataatgcc agcctgttaa acgccgtcga 600

cgagtctaac ggacaccaac cagcgaacca gcagcgtcgc gtcgggccaa gcgaagcaga 660

cggcacggca tctctgtcgc tgcctctgga cccctctcga gagttccgct ccaccgttgg 720

acttgctccg ctgtcggcat ccagaaattg cgtggcggag cggcagacgt gagccggcac 780

ggcaggcggc ctcctcctcc tctcacggca ccggcagcta cgggggattc ctttcccacc 840

gctccttcgc tttcccttcc tcgcccgccg taataaatag acaccccctc cacaccctct 900

ttccccaacc tcgtgttgtt cggagcgcac acacacacaa ccagatctcc cccaaatcca 960

cccgtcggca cctccgcttc aaggtacgcc gctcgtcctc cccccccccc cctctctacc 1020

ttctctagat cggcgttccg gtccatggtt agggcccggt agttctactt ctgttcatgt 1080

ttgtgttaga tccgtgtttg tgttagatcc gtgctgctag cgttcgtaca cggatgcgac 1140

ctgtacgtca gacacgttct gattgctaac ttgccagtgt ttctctttgg ggaatcctgg 1200

gatggctcta gccgttccgc agacgggatc gatttcatga ttttttttgt ttcgttgcat 1260

agggtttggt ttgccctttt cctttatttc aatatatgcc gtgcacttgt ttgtcgggtc 1320

atcttttcat gctttttttt gtcttggttg tgatgatgtg gtctggttgg gcggtcgttc 1380

tagatcggag tagaattctg tttcaaacta cctggtggat ttattaattt tggatctgta 1440

tgtgtgtgcc atacatattc atagttacga attgaagatg atggatggaa atatcgatct 1500

aggataggta tacatgttga tgcgggtttt actgatgcat atacagagat gctttttgtt 1560

cgcttggttg tgatgatgtg gtgtggttgg gcggtcgttc attcgttcta gatcggagta 1620

gaatactgtt tcaaactacc tggtgtattt attaattttg gaactgtatg tgtgtgtcat 1680

acatcttcat agttacgagt ttaagatgga tggaaatatc gatctaggat aggtatacat 1740

gttgatgtgg gttttactga tgcatataca tgatggcata tgcagcatct attcatatgc 1800

tctaaccttg agtacctatc tattataata aacaagtatg ttttataatt attttgatct 1860

tgatatactt ggatgatggc atatgcagca gctatatgtg gattttttta gccctgcctt 1920

catacgctat ttatttgctt ggtactgttt cttttgtcga tgctcaccct gttgtttggt 1980

gttacttgga tccatggagg ctcccccttc tctttccatc gtcgacgaag acggcttcgt 2040

catcgacctc tcgctgacgc tgggcctgac atcgcctccg ccctctccag gcggcgcgtc 2100

tccttccatt cctccgggaa gaggcggcgg cggcggcacg agtggtggtg acaacaacag 2160

aggcagcaga ggtggtggca atggtggtgg tggtggtggt gtgaggctct tcccttgcct 2220

cttctgcaac aagaagtttc tcaagtcgca ggcgctaggg ggacaccaga atgcacacaa 2280

gaaggagagg agcgtcgggt ggaacaccca cctctacctc ccggccggcg tcgccgccgc 2340

cacgacgacg acgacgacga cgacggcgat ggccgtccct gacatggtcg gcatgcccac 2400

ccaccagatg tcctccatgg cgttgcactc ctgccggcct caccagggct cccatgtcac 2460

cgccgccgac atcgccacgc tggctgcgcc gccacattac accgtcgacc atggcgttgc 2520

cggcatcgcc agcggcggtg gtgacagctc ggtggggtgg cgccaaaggc aaagggaggc 2580

cggcggcgag aagcagaggc aggttgatct caacctgaag ctctagggta ccgagctcga 2640

atttccccga tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg 2700

gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca 2760

tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg caattataca 2820

tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg 2880

tgtcatctat gttactagat cgggaattc 2909

Claims

1. The application of the protein OsZFP213 in increasing the salt resistance of rice;

the protein OsZFP213 is a protein shown in a sequence 2 in a sequence table.

2. Use of a nucleic acid molecule encoding the protein OsZFP213 of claim 1 for increasing salt tolerance in rice.

3. method for breeding transgenic plants or two methods for breeding transgenic plants:

the method for cultivating transgenic plant comprises introducing substance for improving expression and/or activity of OsZFP213 as protein in claim 1 into recipient plant to obtain transgenic plant A, wherein the transgenic plant A has improved stress resistance compared with the recipient plant;

the second method for cultivating transgenic plants comprises the step of introducing a substance which inhibits the expression and/or activity of the protein OsZFP213 of claim 1 into a receptor plant to obtain a transgenic plant B; said transgenic plant b has reduced stress resistance as compared to said recipient plant;

the plant is rice;

the stress resistance is salt resistance.

4. Plant breeding method or plant breeding method two:

the plant breeding method , comprising the steps of increasing the content and/or activity of OsZFP213 protein of claim 1 in a plant, thereby improving the stress resistance of the plant;

the second plant breeding method comprises the following steps: reducing the content and/or activity of the protein OsZFP213 of claim 1 in a plant, thereby reducing the stress resistance of the plant;

the plant is rice;

the stress resistance is salt resistance.