CN106520723B

CN106520723B - Protein VvMas, coding gene and application of protein VvMas in improving salt tolerance of plants

Info

Publication number: CN106520723B
Application number: CN201611045870.6A
Authority: CN
Inventors: 王飞兵; 陈新红; 周青
Original assignee: Huaiyin Institute of Technology
Current assignee: Dongdai Jinan Intelligent Technology Co ltd
Priority date: 2016-11-22
Filing date: 2016-11-22
Publication date: 2020-01-21
Anticipated expiration: 2036-11-22
Also published as: CN106520723A

Abstract

The invention provides a protein VvMas, an encoding gene, a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing the encoding gene, and a primer pair for amplifying the full length of the encoding gene or any segment thereof; also provides a protein VvMas, an encoding gene, and application of a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing the encoding gene in improving the salt tolerance of plants. The protein and the coding gene thereof have important application value for cultivating salt-tolerant plant varieties, thereby having important significance for improving the crop yield and having wide application space and market prospect in the agricultural field.

Description

Protein VvMas, coding gene and application of protein VvMas in improving salt tolerance of plants

Technical Field

The invention relates to a plant salt tolerance related protein VvMas and a coding gene and application thereof, in particular to a salt tolerance related protein VvMas from grapes and a coding gene and application thereof.

Background

There is a large area of salinized land in the world. According to statistics, the total of the total weight of the product is 8 hundred million hm all over the world²In the saline-alkali soil, secondary salinized land which occupies 33% of the cultivated area is also arranged in the irrigation area, and the development of modern agriculture is seriously influenced by the salinization of the soil. In China, nearly one tenth of land for secondary salt collapse exists in 18 hundred million acres of cultivated land in China, and 2000 kilohm of land²Barren saltAnd (3) ground. Generally, the salt concentration is 0.2% -0.5% to affect the growth of crops, but the salt content of saline-alkali soil is 0.6% -10% mostly. The existence of large-area salinized land seriously affects the grain production and becomes a main factor for limiting the agricultural production. With the dramatic increase of the population of the world and the annual decline of arable area, the grain production safety is seriously threatened, and the method is increasingly serious for China with relatively small per capita arable area. Through the deep research on the plant salt-tolerant mechanism, the cultivation of new species of salt-tolerant crops is one of the most economic and effective measures for utilizing saline-alkali soil resources.

The salt tolerance mechanism of plants is quite complex, and the salt tolerance mechanism relates to various aspects such as growth and development, morphological structure, physiological characteristics, metabolic regulation and the like. When a plant is stressed by salt, a series of changes occur in aspects of plant morphology, physiology, biochemistry and the like, so that the survival of the plant can be maintained. The salt damage inhibits the growth and differentiation of plant tissues and organs, influences the structure of plant cell membranes, increases the permeability of the cell membranes, reduces the photosynthetic rate, causes the extravasation of a large amount of electrolytes and non-electrolytes, changes the components of membrane lipids, influences the components and activity of membrane proteins and further influences the physiological metabolism of plants. Therefore, a series of mechanisms for resisting external adverse environmental changes are gradually formed in the long-term evolution and adaptation process of the plants.

Under the condition of salt stress, the plant adopts a certain strategy to prevent or reduce the harm of salt, and develops a series of salt tolerance mechanisms in the long-term evolution process. With the rapid development of molecular biology, the physiological biochemical mechanism of plant salt tolerance is increasingly clear, so that the cloning of the plant salt tolerance related gene becomes possible. The research of plant salt tolerance physiology is enhanced, the life activity rule of the plant under adverse circumstances is proved and artificially regulated, and the excellent variety with adverse environment resistance is cultivated, so that the yield and the quality of the crop are improved, and the method has important significance for obtaining agricultural high and stable yield.

Disclosure of Invention

The technical problem is as follows: in order to solve the defects of the prior art, the invention provides a plant salt tolerance related protein VvMas and a coding gene thereof, and also provides application of the protein.

The technical scheme is as follows: the invention provides a protein related to plant salt tolerance, which is named as VvMas (alpha/beta hydrolase) and is derived from grape (vitas vinifera) and is (a) or (b) as follows:

(a) is represented by a sequence SEQ ID NO:2, and 2, or a pharmaceutically acceptable salt thereof;

(b) and (2) mixing the sequence SEQ ID NO:2 and the amino acid residue sequence is subjected to substitution and/or deletion and/or addition of one or more amino acid residues, and is related to plant salt tolerance and is represented by SEQ ID NO:2 derived protein.

The invention also provides a gene for coding the protein VvMas.

The gene, protein related to carbon and nitrogen metabolism, is the gene described in any one of the following (1) to (3):

(1) the nucleotide sequence is SEQ ID NO: 1;

(2) a DNA molecule which is hybridized with the DNA sequence defined in the step (1) under strict conditions and codes the plant salt tolerance related protein;

(3) a DNA molecule which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology with the DNA sequence defined in (1) or (2) and encodes a plant salt tolerance-related protein.

The stringent conditions may be hybridization with a solution of 6 XSSC, 0.5% SDS at 65 ℃ followed by washing the membrane once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

Sequence SEQ ID NO: 1 consists of 1224 bases and encodes the sequence of SEQ ID NO: 2.

The invention also provides an expression cassette, a recombinant expression vector, a transgenic cell line or a recombinant bacterium containing the coding gene of the protein related to the plant salt tolerance.

The recombinant expression vector is obtained by inserting the gene into an expression vector.

Specifically, the recombinant vector is obtained by inserting the coding gene between multiple cloning sites of the vector pCBGUS.

Wherein, the vector pCBGUS is obtained by a method comprising the following steps:

(1) carrying out double enzyme digestion on the pCAMBIA1301 vector by Hind III and EcoR I, and recovering a large fragment of the vector;

(2) carrying out double enzyme digestion on the pBI 121 vector by Hind III and EcoR I, and recovering a fragment containing the gusA gene;

(3) and (3) connecting the large vector fragment recovered in the step (1) with the fragment containing the gusA gene recovered in the step (2) to obtain the vector pCBGUS.

The pCAMBIA1301 vector is purchased from CAMBIA corporation; the pBI 121 vector was purchased from Clontech.

The invention also provides a primer pair for amplifying the full length of the coding gene of the protein related to the salt tolerance of the plant or any fragment thereof.

Specifically, the sequences of the primer pairs are as follows:

GSP-1：5’-CTGAGACGAGTTGGGGTGGAA-3’

GSP-2：5’-GTTTTCCCAGTCACGAC-3’

GSP-3：5’-GACTTGGCCTCCAGGTTGACCTTGA-3’

GSP-4：5’-GGCCACGCGTCGACTAGTACGGGGGGGGGG-3’

GSP-5：5’-ATGAAAGGCGTCTTTTCGGCGCCAG-3’

GSP-6：5’-TTACACAAGACATCTACTTTTCCAA-3’

the invention also provides the application of the protein VvMas, the coding gene thereof or the recombinant vector, the expression cassette, the transgenic cell line and the recombinant bacteria thereof in improving the salt tolerance of plants; the plant is a dicotyledonous plant or a monocotyledonous plant.

The invention also provides a method for cultivating the transgenic plant, which comprises the following steps: introducing a gene encoding the protein of claim 1 into a target plant.

Specifically, the gene encoding the protein of claim 1 is introduced into a target plant by the recombinant vector of

claim

4 or 5; the plant is particularly a dicotyledonous plant or a monocotyledonous plant.

The target plant is a dicotyledonous plant or a monocotyledonous plant; the dicotyledonous plant is preferably Arabidopsis thaliana or rice.

Has the advantages that: the protein coded by the VvMas gene provided by the invention can improve the salt tolerance of plants, has important application value and provides important basis for the research of improving the salt tolerance of grapes.

The protein and the coding gene thereof have important application value for cultivating salt-tolerant plant varieties, thereby having important significance for improving the crop yield and having wide application space and market prospect in the agricultural field.

Drawings

FIG. 1 is a diagram of a plant expression vector of the grape VvMas gene of the present invention.

FIG. 2 is a diagram showing the result of PCR detection of VvMas transgenic Arabidopsis plants of the present invention.

FIG. 3 is a diagram showing the result of PCR detection of VvMas transgenic rice plant of the present invention.

FIG. 4 is a salt tolerant potted plant identification map of a VvMas transgenic Arabidopsis plant of the invention.

FIG. 5 is a salt tolerance in vitro identification chart of VvMas transgenic rice plant of the present invention.

FIG. 6 is a salt tolerance hydroponic identification chart of VvMas transgenic rice plants of the present invention.

FIG. 7 is a salt tolerant potted plant identification map of a VvMas transgenic rice plant of the invention.

Detailed Description

In the following examples, the test materials and sources used include:

grape (Vitis vinifera) variety PN40024 is preserved in laboratories of plant production and processing practice education centers of Jiangsu province of the Huaiyin institute of Industrial science and food engineering.

Seeds of Arabidopsis thaliana (Arabidopsis thaliana) were passed through 2.5% (v/v) CaClO₂Planting in black soil after disinfection: vermiculite: in the mixed matrix of perlite (1:1:1), the mixture is cultured for 16h under illumination (16h under illumination, 8h under dark condition and cold light source) at 22 ℃ for 2 weeks.

No. 11 of the rice (Oryza sativa) variety, which is preserved in laboratories of the plant production and processing practice education center of Jiangsu province of the Huaiyin institute of Industrial and science and food engineering.

Escherichia coli (Escherichia coli) DH5 alpha was stored in laboratories of the education center for plant production and processing practice in Jiangsu province of the institute of food engineering and Life sciences of Huaiyin institute of Industrial science. Cloning vector PMD-18-Simple T, various restriction enzymes, Taq polymerase, ligase, dNTP, 10 XPCR buffer and DNA marker were purchased from Bao bioengineering Dai Lian Limited. All chemicals were purchased from sigma chemical company, usa and from pharmaceutical chemicals, shanghai.

For general Molecular biology procedures of the present invention, reference is made in detail to Molecular cloning, 2nd, Cold Spring Harbor Laboratory Press, 1989.

Conventional genetic manipulations in the examples described below were performed with reference to the Molecular cloning literature [ Sambook J, fress EF, Manndes T et al in: Molecular cloning.2nd. Cold Spring Harbor laboratory Press,1989 ].

Example 1 obtaining of a protein related to salt tolerance of grape and a Gene encoding the same

1. Experimental Material

Unfolding leaves and leaves of the grape variety PN40024 aseptic seedlings, taking down the aseptic seedlings, quickly freezing the aseptic seedlings by liquid nitrogen, and preserving the aseptic seedlings at minus 80 ℃.

2. Leaf Total RNA extraction and purification

Taking about 2.0g of leaves of PN40024 aseptic seedlings, grinding the leaves into powder in liquid nitrogen, adding the powder into a10 mL centrifuge tube, and extracting the total RNA of the tuberous roots of the sweet potatoes by using an Aplygen plant RNA extraction kit (Aplygen Technologies Inc, Beijing), wherein the kit comprises: plant RNA Reagent, Plant tissue cracking, RNA separation, removal of Plant polysaccharides and polyphenols; extracting Reagent, and organically extracting to remove protein, DNA, polysaccharide and polyphenol; plant RNA Aid, removing Plant polysaccharide polyphenols and secondary metabolites. mRNA was purified from total RNA using the QIAGEN Oligotex Mini mRNA Kit (QIAGEN, GmbH, Germany). And finally, taking 1 mu L of the total RNA to be subjected to 1.2% agarose gel electrophoresis for detecting the integrity of the total RNA, taking another 2 mu L of the total RNA to be diluted to 500 mu L, detecting the quality (OD260) and the purity (OD260/OD280) of the total RNA by using an ultraviolet spectrophotometer, and extracting the total RNA of leaves of the PN40024 sterile seedlings, wherein the total RNA is detected by using non-denaturing gel agarose gel electrophoresis, the bands of 28S and 18S are clear, the brightness ratio of the two is 1.5-2: 1, the total RNA is not degraded, the purified mRNA meets the experimental requirements, and the mRNA can be used for cloning the full length of the VvMas protein cDNA.

Full Length cloning of VvMas protein cDNA

Full-length cloning of VvMas protein cDNA was performed using VvMas EST fragment design primers obtained in this laboratory.

(1)3’-RACE

PCR was performed using the VvMas EST forward primer GSP-1 and reverse primer GSP-2 using PN40024cDNA as a template. The primer sequences are as follows:

GSP-1：5’-CTGAGACGAGTTGGGGTGGAA-3’

GSP-2：5’-GTTTTCCCAGTCACGAC-3’

the 3' RACE fragment obtained by PCR was recovered and ligated with pMD19-T vector (purchased from Liuhe TongO Co., Ltd., Beijing, catalog No. D102A) for TA cloning, and sequenced with BcaBESTTM Sequencing Primers/M13 Primers.

(2)5’-RACE

PCR was performed using the PN40024cDNA as a template and VvMas EST as a forward primer GSP-3 and a reverse primer GSP-4. The primer sequences are as follows:

GSP-3：5’-GACTTGGCCTCCAGGTTGACCTTGA-3’

GSP-4：5’-GGCCACGCGTCGACTAGTACGGGGGGGGGG-3’

the 5' RACE fragment obtained by PCR was recovered and ligated with pMD19-T vector (purchased from Liuhe TongO Co., Ltd., Beijing, catalog No. D102A) for TA cloning, and sequenced with BcaBESTTM Sequencing Primers/M13 Primers.

(3) PCR amplification of coding region of VvMas protein cDNA

Candidate grape VvMas protein cDNA sequences were spliced using DNAMAN 7.0 software. The coding region of VvMas protein cDNA was further amplified by PCR using forward primer GSP-5 and reverse primer GSP-6. The primer sequences are as follows:

GSP-5：5’-ATGAAAGGCGTCTTTTCGGCGCCAG-3’

GSP-6：5’-TTACACAAGACATCTACTTTTCCAA-3’

PN40024 leaf total RNA is used as a template through oligo (dT) reverse transcription, and PCR amplification is carried out by using high fidelity Fastpfu enzyme under the PCR condition of 95 ℃ for 1min, then 40 cycles are carried out at 95 ℃ for 20s, 53 ℃ for 20s and 72 ℃ for 1min, and finally extension is carried out at 72 ℃ for 10 min. Detecting the PCR amplification product by agarose gel electrophoresis to obtain an amplification fragment with the length of 1224 bp.

And (3) synthesizing the results of the steps to obtain a target cDNA sequence, wherein the nucleotide sequence of the target cDNA sequence is shown as a sequence SEQID NO: 1 is shown. Sequence SEQ ID NO: 1 consists of 1224 bases, the 1 st to 1224 st bases from the 5' end are an open reading frame of the base, and the base encodes a polypeptide with a sequence SEQ ID NO:2, or a pharmaceutically acceptable salt thereof. Sequence SEQ ID NO:2 consists of 407 amino acid residues. The gene is named as VvMas, and the protein coded by the gene is named as VvMas.

Example 2 construction of VvMas Gene overexpression vector

The sequences identified in example 1 were sequenced to identify the correct sequence containing SEQ ID NO: 1, double-digesting the DNA fragment with BamH I and Sac I, recovering the DNA fragment with 1% agarose gel, and passing through T₄And (3) connecting the recovered VvMas gene fragment with a plasmid pYPx245 containing a double 35S promoter by using DNA ligase, and performing enzyme digestion identification and sequence analysis and determination to obtain a recombinant plasmid AH128 containing the grape VvMas gene. The expression vector also contains gusA reporter gene and intron kanamycin resistance marker gene, and is shown in FIG. 1.

Example 3 transformation of Arabidopsis thaliana with VvMas Gene

The plant expression vector pCAMBIA1301-VvMas of the grape VvMas gene constructed in the embodiment 2 is used for transforming arabidopsis thaliana by a dipping method, and the specific method is as follows:

1. preparation of Agrobacterium

(1) Agrobacterium tumefaciens strain LBA4404 (BiovectorCo., LTD) was transformed with pCAMBIA1301-VvMas by an electric shock method to obtain recombinant Agrobacterium containing pCAMBIA1301-VvMas, which was plated on a plate containing kanamycin resistance to select transformants.

(2) A single strain of Agrobacterium was inoculated into 5mL of LB liquid medium (rifampicin 50. mu.g/mL, chloramphenicol 100. mu.g/mL) and cultured at 28 ℃ and 250rpm for 20 hours.

(3) 1mL of the bacterial suspension was transferred to 20-30mL of LB liquid medium (rifampicin 50. mu.g/mL, chloramphenicol 100. mu.g/mL), cultured at 28 ℃ and 250rpm for about 12 hours, and the OD 600 was determined to be approximately equal to 1.5.

(4) The cells were collected by centrifugation at 8000rpm, 4 ℃ for 10min, resuspended in Agrobacterium transformation permeate (5% sucrose, 0.05% Silwet L-77) and diluted to OD 600. apprxeq.0.8.

2. Transformation of Arabidopsis by flower dipping method

(1) Soaking the flower bolt of the arabidopsis into the staining solution, slightly stirring for about 10s, taking out, covering the arabidopsis with a freshness protection package after all transformation is finished so as to keep a humid environment, horizontally placing, culturing at 22 ℃ in a dark place, and removing the freshness protection package after 24h for upright culture.

(2) After four days of the primary transformation, the transformation can be carried out again and repeated twice, and the transformation is carried out three times in total, so that the buds of different periods developing on the inflorescence can be transformed, and the transformation efficiency is improved.

(3) After about two months of growth, the seeds were collected and stored in a refrigerator at 4 ℃ for future use.

Arabidopsis transformed by the dipping method normally blooms and forms a seed about two months later.

Example 3VvMas Gene transfer to Rice

The plant expression vector pCAMBIA1301-VvMas of the grape VvMas gene constructed in the embodiment 2 is used for transforming rice, and the specific method is as follows:

1. preparation of Agrobacterium

(1) The pCAMBIA1301-VvMas was used to transform Agrobacterium tumefaciens EHA105 strain (Biovector Co., LTD) by an electric shock method to obtain recombinant Agrobacterium containing pCAMBIA1301-VvMas, which was plated on a plate containing kanamycin resistance to select transformants.

2. Obtaining mature embryo callus of rice

(1) Removing glumes of No. 11 seeds of the mature rice variety, and disinfecting for 1-2min by using 70% alcohol;

(2) then soaking with 20% sodium hypochlorite for 30-40min, washing with sterile distilled water for 4 times, transferring the seeds onto sterilized filter paper, blotting surface water, and inoculating on NB induction culture medium;

(3) after dark culture for 7-10 days, when scutellum is enlarged and endosperm is softened, embryo and bud are removed, and the peeled embryogenic callus is transferred to NB subculture medium for about 3w subculture once, and can be used as a receptor for transformation after 2-3 subcultures.

3. Agrobacterium-mediated transformation of rice callus

(1) Selecting good embryogenic callus, and soaking in the staining solution for 30 min;

(2) taking out the callus, removing the redundant bacteria liquid by using sterile filter paper, and then placing the callus on an NB co-culture medium for culturing until colonies just appear (about 2-3 d);

(3) shaking and washing with sterile water for 3-4 times until the supernatant is completely clean, and shaking and washing with 500mg/L cefmenomycin solution for 40 min;

(4) taking out the callus, putting the callus into a sterile culture dish only provided with filter paper, air-drying the callus for 4 hours at 0.4m/s, and transferring the callus into an NB screening culture medium for two screening rounds (each round is 3-4 w);

(5) pre-differentiating the resistant callus for 2-3w, and then transferring the resistant callus to a differentiation medium for 2-3w of illumination culture;

(6) when the sprouts grow to about 1cm, transferring the sprouts into a strong seedling culture medium for about 30d of culture;

(7) removing the sealing film, hardening off the seedling, culturing for about one week, and transplanting into soil.

Example 4 VvMas Gene transgenic Arabidopsis plants PCR assay

1. Screening of transgenic Arabidopsis seeds

(1) Weighing 25-30mg of seeds and putting the seeds into a 1.5mL centrifuge tube;

(2) sterilizing with 1mL of 75% ethanol for 1min (shaking and oscillating continuously), centrifuging at 8000rpm for 5s, and removing supernatant;

(3) adding 1mL of filtered bleaching powder (2.5%), sterilizing for 15min (shaking continuously, fully sterilizing), centrifuging at 8000rpm for 5s, and removing supernatant;

(4) washing with sterile water for 3-4 times;

(5) the seeds were evenly spread on 1/2MS plates (hygromycin 50. mu.g/mL), sealed with Parafilm, placed in a refrigerator at 4 ℃ for two days, and cultured at 22 ℃ for 16h under light for 10 days.

(6) Transplanting the resistant plant into a pot for culture, detecting GUS activity after the seedling is bigger, and selecting a positive plant (T)₁) Culturing until blooming and fructification, collecting T₁T knot on the plant₂Seeds, further screening to obtain T₃And (4) seeds.

2. PCR detection of transgenic Arabidopsis plants

(1) Test method

Extraction of T by CTAB method₃Genomic DNA of Arabidopsis transgenic plants and wild-type plants. PCR detection is carried out by a conventional method, and VvMas gene primers used are as follows: primer 1: 5'-ACAGCGTCTCCGACCTGATGCA-3' and primer 2: 5'-AGTCAATGACCGCTGTTATGCG-3' are provided. To a 0.2mL Eppendorf centrifuge tube were added 2. mu.L of 10 XPCR buffer, 1. mu.L of 4dNTP (10mol/L), 1. mu.L of each primer (10. mu. mol/L), 0.25. mu.L of template DNA (50ng/uL) 2. mu. L, TaqDNA polymerase, and ddH₂O to a total volume of 20. mu.L. The reaction program is pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, renaturation at 55 ℃ for 30s, and extension at 72 ℃ for 2min, and 35 cycles are total.

(2) Test results

The results of the electrophoretic detection amplification are shown in FIG. 2 (in FIG. 2, lane M is marker, lane W is water, lane P is a positive control (recombinant plasmid pCAMBIA1301-VvMas), lane Col-0 is a wild type Arabidopsis plant, and lanes #1- #5 are Arabidopsis transgenic plants transformed with pCAMBIA 1301-VvMas). As can be seen from the figure, 591bp of target bands are amplified by an arabidopsis thaliana pseudotransgenic plant transformed with pCAMBIA1301-VvMas and a positive control, which shows that the VvMas gene is integrated into the genome of arabidopsis thaliana, and the regenerated plants are proved to be transgenic plants; the target band of 591bp is not amplified from the wild type arabidopsis thaliana plant. Transgenic plants were subsequently analyzed for function.

Example 5 VvMas Gene transgenic Rice plant PCR assay

(1) Test method

Extraction of T by CTAB method₂Genomic DNA of rice transgenic plants and wild-type plants. PCR detection is carried out by a conventional method, and VvMas gene primers used are as follows: primer 1: 5'-ACAGCGTCTCCGACCTGATGCA-3' and primer 2: 5'-AGTCAATGACCGCTGTTATGCG-3' are provided. To a 0.2mL Eppendorf centrifuge tube were added 2. mu.L of 10 XPCR buffer, 1. mu.L of 4dNTP (10mol/L), 1. mu.L of each primer (10. mu. mol/L), 2. mu.L of template DNA (50ng/uL), 0.25. mu.L of Taq DNA polymerase, and ddH₂O to a total volume of 20. mu.L. The reaction program is pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, renaturation at 55 ℃ for 30s, and extension at 72 ℃ for 2min, and 35 cycles are total.

(2) Test results

The results of the electrophoresis are shown in FIG. 3 (in FIG. 3, lane M is marker, lane W is water, lane P is a positive control (recombinant plasmid pCAMBIA1301-VvMas), lane WT is a wild-type rice plant, and lane OE1-OE11 is a transgenic rice plant transformed with pCAMBIA 1301-VvMas). As can be seen from the figure, the 591bp target band is amplified by the rice pseudotransgenic plant transformed with pCAMBIA1301-VvMas and the positive control, which shows that the VvMas gene is integrated into the genome of rice, and the regenerated plants are proved to be transgenic plants; the 591bp target band is not amplified from the wild rice plant. Transgenic plants were subsequently analyzed for function.

Example 6 identification of salt tolerance of VvMas Gene transgenic Arabidopsis plants

(1) Test method

After 2w of transgenic arabidopsis and wild type seeds are cultured on 1/2MS culture medium, the plants are transplanted into pots for 2w of culture, and then salt stress treatment is carried out. The plants were treated with 4w of 1/2 Hoagland nutrient solution containing 300mM NaCl by irrigating them 1 time per 2d, 200mL each, and photographing and counting the survival rate.

(2) Test results

The results show that the growth state of the transgenic plants is obviously better than that of the wild plants and the survival rate of the transgenic plants is obviously higher than that of the wild plants after 4w of salt treatment through the identification of the salt-tolerant pot plants as shown in figure 4. The salt tolerance of transgenic arabidopsis plants is obviously improved by over-expressing the VvMas gene.

Example 7 identification of salt tolerance of VvMas Gene transgenic Rice plants

1. Comparison of growth potential of transgenic Rice plants

(1) Test method

Sterilizing the seeds of the transgenic rice material and the wild type material, sowing the seeds on an MS solid flat plate, selecting the seeds with consistent germination states after the seeds germinate for 2-3d, respectively sowing the seeds on different medium finger tube culture media of MS and MS + NaCl (200mM), and after the seedlings grow for 7-10d, carrying out photographing and growth vigor statistics on the difference of the growth vigor of the seedlings treated differently, wherein the statistics comprise the seedling length and fresh weight data.

(2) Test results

The results show that under the salt stress treatment conditions, the results are shown in FIG. 5, and the transgenic material and the wild type WT material are both reduced in size due to the salt stress conditions; however, compared with wild WT material, the growth state of the transgenic material is relatively better, and the statistics of growth potential data show that the seedling length and fresh weight of the transgenic material are better than those of the wild WT material.

2. Salt tolerance water culture identification of transgenic rice plant

(1) Test method

Will be homozygous T₂Transgenic and wild rice seeds were inoculated on MS medium and grown for approximately 3-4 days. Selecting seedlings with consistent growth vigor, transplanting the seedlings into a 96-hole PCR plate (the bottom of the PCR plate is cut off), and replacing the nutrient solution every 1d to avoid the growth of bacteria at the roots of the seedlings, wherein the seedlings grow normally for 4 w; it was transferred to 200mM NaCl Hoagland solution for stress treatment for 2w, its phenotype was observed, and it was subjected to a photograph and investigated for its survival rate.

(2) Test results

The results show that after the salt stress treatment condition of 2w, the results are shown in fig. 6, the difference of the transgenic material is obvious compared with the wild type material, the growth state of the transgenic material is obviously better than that of the wild type material, and the survival rate of the transgenic plant is obviously higher than that of the wild plant. The salt tolerance of transgenic rice plants is obviously improved by over-expressing the VvMas gene.

3. Transgenic rice plant salt tolerance potted plant identification

(1) Test method

To further verify the salt tolerance of the transgenic rice material, homozygous T's were used₂Sterilizing the surfaces of the transgenic rice and wild rice seeds, accelerating germination by using purified water, inoculating the seeds on an MS culture medium, and growing for about 3-4 d. Selecting seedlings with consistent growth vigor, and planting the seedlings in nutrient soil: in the nutrient soil with 1:2 vermiculite, watering is carried out every day, and salt stress treatment is started when the plants grow to 4 true leaves. 2w were treated by irrigating 1 time per 2d with 1/2 Hoagland nutrient solution containing 200mM NaCl, 200mL each, and the phenotype was observed, followed by photographing and investigation of the survival rate.

(2) Test results

The results of fig. 7 show that after the salt stress treatment condition is 2w, the salt-tolerant potting experiment shows that the difference of the transgenic material is obvious compared with the wild type material, the growth state of the transgenic material is obviously superior to that of the wild type material, and the survival rate of the transgenic plant is obviously higher than that of the wild type plant. The salt tolerance of transgenic rice plants is obviously improved by over-expressing the VvMas gene.

<110> Huaiyin institute of Industrial and research

<120> protein VvMas, coding gene and application thereof in improving salt tolerance of plants

<130>20161118002

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<170>PatentIn version 3.3

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gtcaagggta tctcaaagga tggtagtggt gggagttcta gtgaaaagaa tgatgaaaga 900

gaagattcag atgttcctcc taaggacaat gggggaagtc ctgaaagccc ttccactgaa 960

acccaattgc cagaggctcc agaaagttcg ggttctcata gcttagatga ccagctcctc 1020

agcaatgcaa aggtttgctt caccagtcct gaacatgtga ggcttcccat atctcatgca 1080

ttttttgaga aacagcaaga cattgtatcc acaaggtttg tccaaccggc ttgggagatt 1140

tttattcttt gtctgcttcc tctctatgtg gaaacaatgt acattacttg gattttttgt 1200

tggaaaagta gatgtcttgt gtaa 1224

<210>2

<211>407

<212>PRT

<213>Vitis vinifera

<220>

<221>MISC_FEATURE

<222>(1)..(407)

<400>2

Met Lys Gly Val Phe Ser Ala Pro Gly Asp Tyr Ile His Phe Lys Ser

1 5 10 15

Gln Val Pro Leu His Lys Ile Pro Ile Gly Thr Lys Gln Trp Arg Tyr

20 25 30

Tyr Asp Phe Gly Pro Lys Val Val Pro Pro Leu Ile Cys Leu Pro Gly

35 40 45

Thr Ala Gly Thr Ala Asp Val Tyr Tyr Lys Gln Ile Met Ser Leu Ser

50 55 60

Ile Lys Gly Tyr Arg Val Ile Ser Val Asp Ile Pro Arg Val Trp Asn

65 70 75 80

His His Glu Trp Ile Gln Ala Phe Glu Lys Phe Leu Asp Ala Ile Asp

85 90 95

Val His His Ile His Leu Tyr Gly Thr Ser Leu Gly Gly Phe Leu Ala

100 105 110

Gln Leu Phe Ala Gln His Arg Pro Arg Arg Val Arg Ser Leu Ile Leu

115 120 125

Ser Asn Ser Phe Leu Glu Thr Arg Ser Phe Ser Ser Ala Met Pro Trp

130 135 140

Ala Pro Ile Val Ser Trp Thr Pro Ser Phe Leu Leu Lys Arg Tyr Val

145 150 155 160

Leu Thr Gly Ile Pro Asp Gly Pro His Glu Pro Phe Ile Ala Asp Ser

165 170 175

Val Asp Phe Val Val Ser Gln Val Glu Thr Leu Ser Arg Glu Asp Leu

180 185 190

Ala Ser Arg Leu Thr Leu Thr Val Asp Ala Ala Ser Ile Gly Pro Leu

195 200 205

Leu Leu Ser Asp Ser Phe Ile Thr Leu Met Asp Thr Asn Asp Tyr Cys

210 215 220

Ser Ile Pro Gln Gln Leu Lys Asp Gln Leu Ser Glu Arg Tyr Pro Gly

225 230 235 240

Ala Arg Arg Ala Tyr Leu Lys Thr Gly Gly Asp Phe Pro Phe Leu Ser

245 250 255

Arg Ser Asp Glu Val Asn Leu His Leu Gln Leu His Leu Arg Arg Val

260 265 270

Gly Val Glu Ala Arg Pro Asp Leu Val Lys Gly Ile Ser Lys Asp Gly

275 280 285

Ser Gly Gly Ser Ser Ser Glu Lys Asn Asp Glu Arg Glu Asp Ser Asp

290 295 300

Val Pro Pro Lys Asp Asn Gly Gly Ser Pro Glu Ser Pro Ser Thr Glu

305 310 315 320

Thr Gln Leu Pro Glu Ala Pro Glu Ser Ser Gly Ser His Ser Leu Asp

325 330 335

Asp Gln Leu Leu Ser Asn Ala Lys Val Cys Phe Thr Ser Pro Glu His

340 345 350

Val Arg Leu Pro Ile Ser His Ala Phe Phe Glu Lys Gln Gln Asp Ile

355 360 365

Val Ser Thr Arg Phe Val Gln Pro Ala Trp Glu Ile Phe Ile Leu Cys

370 375 380

Leu Leu Pro Leu Tyr Val Glu Thr Met Tyr Ile Thr Trp Ile Phe Cys

385 390 395 400

Trp Lys Ser Arg Cys Leu Val

405

<210>3

<211>21

<212>DNA

<213>Artificial

<220>

<223>GSP-1

<220>

<221>misc_feature

<222>(1)..(21)

<400>3

ctgagacgag ttggggtgga a 21

<210>4

<211>17

<212>DNA

<213>Artificial

<220>

<223>GSP-2

<220>

<221>misc_feature

<222>(1)..(17)

<400>4

gttttcccag tcacgac 17

<210>5

<211>25

<212>DNA

<213>Artificial

<220>

<223>GSP-3

<220>

<221>misc_feature

<222>(1)..(25)

<400>5

gacttggcct ccaggttgac cttga 25

<210>6

<211>30

<212>DNA

<213>Artificial

<220>

<223>GSP-4

<220>

<221>misc_feature

<222>(1)..(30)

<400>6

ggccacgcgt cgactagtac gggggggggg 30

<210>7

<211>25

<212>DNA

<213>Artificial

<220>

<223>GSP-5

<220>

<221>misc_feature

<222>(1)..(25)

<400>7

atgaaaggcg tcttttcggc gccag 25

<210>8

<211>25

<212>DNA

<213>Artificial

<220>

<223>GSP-6

<220>

<221>misc_feature

<222>(1)..(25)

<400>8

ttacacaaga catctacttt tccaa 25

Claims

1. A method for improving the salt tolerance of plants is characterized in that: protein VvMas, coding gene, recombinant vector, expression cassette or recombinant bacteria are used;

the protein VvMas is a protein consisting of an amino acid sequence shown by SEQ ID NO. 2 in a sequence table;

the coding gene is a coding gene of VvMas protein and is SEQ ID NO: 1;

the recombinant vector, the expression cassette and the recombinant bacteria are obtained by inserting the coding gene of the protein VvMas into the expression vector;

the plants are Arabidopsis thaliana and rice.

2. A method for breeding salt-tolerant transgenic plants, which is characterized by comprising the following steps: the method comprises the following steps: introducing a coding gene of the protein VvMas into a target plant to obtain the gene;

the coding gene of the protein VvMas is SEQ ID NO: 1.

3. The method according to claim 2, wherein the gene coding for the protein VvMas is introduced into the target plant by a recombinant vector containing the gene coding for the protein VvMas; the plants are Arabidopsis thaliana and rice.