CN106146637B

CN106146637B - GmSLT protein for improving salt tolerance of plants, nucleic acid and application

Info

Publication number: CN106146637B
Application number: CN201610756269.1A
Authority: CN
Inventors: 马伟; 刘晓丽; 向东; 厉建蕾; 李泉; 李晴; 熊新彩; 白林泉; 邓子新
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2016-08-29
Filing date: 2016-08-29
Publication date: 2020-11-06
Anticipated expiration: 2036-08-29
Also published as: CN106146637A

Abstract

The invention relates to a GmSLT protein and nucleic acid for improving the salt tolerance of plants in the technical field of botany and application thereof; the GmSLT protein contains one or more of the following amino acid sequences: a domain ABS sequence as shown in SEQ ID NO.3, a TM sequence as shown in SEQ ID NO.4, and a ZBS sequence as shown in SEQ ID NO. 5; the invention also relates to a nucleic acid sequence for coding the GmSLT protein and application of the nucleic acid sequence in enhancing the salt tolerance of plants. According to the invention, the salt-tolerant gene GmSLT is obtained through bioinformatics and gene space-time expression analysis; the salt tolerance of the yeast can be obviously improved by transforming the gene into the yeast; the salt tolerance of corresponding plants can be enhanced by transforming arabidopsis thaliana and rice; the gene of the invention can be used for improving plants and improving the salt tolerance of the plants.

Description

GmSLT protein for improving salt tolerance of plants, nucleic acid and application

Technical Field

The invention belongs to the technical field of botany, and particularly relates to a GmSLT protein for improving salt tolerance of plants, nucleic acid and application thereof.

Background

Salt damage is one of the main causes of crop yield reduction and cultivation area reduction. Therefore, the salt tolerance of crops is improved, and the method makes positive contribution to sustainable development of agricultural economy in China. The important method for solving the problem is to culture a new variety of excellent salt-tolerant products by biotechnology, wherein the essential method is to utilize the salt-tolerant gene resource of plants.

When the plant is stressed by the adversity, a strict structure is formed in time and space, and a coordinated complex network is formed by regulation and control means such as the transcription level, the post-transcription level, the protein level and the like of gene expression. The genes do not play a role in the salt stress response in isolation, but play a role in coordination through the integration of a signal network, so that the plants keep the ion balance in cells and the whole plant body under the salt stress; keeping the water and osmotic balance between cells and the environment and among the cells; maintaining the structure and biological function of protein and nucleic acid biomacromolecule; keep physiological metabolism and energy metabolism balance.

Research shows that the salt tolerance of plants is a quantitative character controlled by multiple genes and is very complex. The plants can resist adverse salinization environment through regulation and control of plant overall physiology, metabolism, gene expression and the like. It has been reported that when some genes of sodium/hydrogen cation transporters, transcription factors, osmotic agent compositions, etc. are transferred into plants and over-expressed, the salt tolerance of these transgenic plants is improved to various degrees. The fact shows that the related gene plays a key role in the aspect of plant salt tolerance. Therefore, it is necessary to research the gene closely related to plant salt tolerance and transfer the gene into plants, so as to improve the salt tolerance of the plants.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a GmSLT protein for improving the salt tolerance of plants, a nucleic acid and an application thereof. The gene of the invention can be used for improving plants and improving the salt tolerance of the plants.

In a first aspect, the invention relates to a GmSLT protein for improving the salt tolerance of plants, wherein the protein comprises one or more of the following amino acid sequences: a structural domain ABS sequence shown as SEQ ID NO.3, a TM sequence shown as SEQ ID NO.4 and a ZBS sequence shown as SEQ ID NO. 5.

Preferably, the protein comprises an amino acid sequence as shown in SEQ ID NO 2.

Preferably, the sequence of the protein is shown as SEQ ID NO. 2.

In a second aspect, the present invention relates to a nucleic acid sequence encoding the aforementioned GmSLT protein.

Preferably, the nucleic acid sequence is shown as SEQ ID NO. 1.

In a third aspect, the invention also relates to the application of the nucleic acid sequence in enhancing the salt tolerance of plants.

In a fourth aspect, the present invention relates to a recombinant expression vector comprising the aforementioned nucleic acid sequence.

In a fifth aspect, the present invention relates to a transgenic cell line comprising the aforementioned nucleic acid sequence.

In a fifth aspect, the present invention relates to a recombinant strain comprising the aforementioned nucleic acid sequence.

In a seventh aspect, the present invention also relates to a method for improving the salt tolerance of plants, comprising the following steps: the nucleic acid sequence is introduced into plant and cultivated to obtain transgenic plant with salt tolerance.

Preferably, the plant is a monocot, a dicot, or a gymnosperm.

Preferably, the plant is a crop, a floral plant, or a forestry plant.

Preferably, the plant is soybean, rice, arabidopsis, wheat, corn, cotton, canola, sorghum, or potato.

In an eighth aspect, the present invention also relates to a method for cultivating salt-tolerant plants, which is characterized by comprising the following steps: and (3) crossing the transgenic plant obtained by the method with a target plant to further obtain a salt-tolerant plant and a filial generation.

In a ninth aspect, the invention also relates to a structural domain sequence for improving the salt tolerance of plants, wherein the structural domain sequence is a structural domain ABS sequence shown as SEQ ID NO.3, a TM sequence shown as SEQ ID NO.4 or a ZBS sequence shown as SEQ ID NO. 5.

The invention has the following beneficial effects: according to the invention, a salt-tolerant gene named GmSLT is obtained through bioinformatics and gene space-time expression analysis; the salt tolerance of the yeast can be obviously improved by transforming the gene into the yeast; the salt tolerance of corresponding plants can be enhanced by transforming arabidopsis thaliana and rice; the gene of the invention can be used for improving plants and improving the salt tolerance of the plants.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1: schematic diagram of GmSLT functional domain;

FIG. 2: GmSLT expression is subjected to a salt tolerance spot test on yeast W303-1 a;

FIG. 3: comparing the growth rate of the yeast overexpressing GmSLT under the salt stress condition with that of a wild type yeast;

FIG. 4: the function of the key structural domain of the GmSLT gene in salt tolerance;

FIG. 5: detecting a GmSLT gene of a To-generation seedling of the GmSLT transgenic rice;

wherein WT is a parent, NTC1 and NTC2 are blank controls, P1 and P2 are positive controls;

FIG. 6: detecting the expression of a part of GmSLT gene transferred rice plant GmSLT gene;

FIG. 7: and (3) a 1# strain T1 generation seedling salt tolerance test result chart of the GmSLT transgenic rice.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as molecular cloning in Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations.

In the examples of the present invention, unless otherwise specified, all experimental methods used were conventional methods.

The materials, reagents, consumables and the like used in the examples of the present invention are commercially available unless otherwise specified.

The present invention is not particularly limited with respect to the plant suitable for use in the present invention, as long as it is suitable for conducting gene transformation operations, such as various crops, flower plants, forestry plants, or the like. The plant may be, for example and without limitation, a dicot, monocot, or gymnosperm. More specifically, the plants include (but are not limited to): rice, arabidopsis, wheat, corn, soybean, cotton, potato, rape, etc.

Example 1 cloning of candidate Soybean genes

Through bioinformatics analysis, some soybean salt-tolerant candidate genes are obtained through prediction. Soybean Hefeng 39 seedlings (reference literature- -Si Zheng Feng, Xia Da Liang, Wang Bao Feng, Bai He Yin, Shi Zui, 2001. Soybean variety-Hefeng 39., China agricultural technology promotion, 2001(4):33-33) were treated with 150mM sodium chloride solution, and then the time-space expression pattern analysis was performed on these candidate genes by using real-time fluorescence quantitative PCR, and it was found that there were a plurality of candidate genes with obvious changes after salt stress. Wherein GmSLT is a soybean salt-tolerant gene obtained by prediction by the method, the soybean seedling root and leaf gene space-time expression pattern analysis is carried out, the expression is obviously up-regulated after 150mM salt stress, the soybean salt-tolerant related gene is presumed, and the gene is cloned;

based on the sequence of the predicted gene GmSLT, a pair of primers was designed using software Primer Premier 5 and amplified using high fidelity enzyme KOD. The amplification template is to obtain cDNA of soybean (RNA is extracted from root and leaf of soybean seedling after salt stress and then is reversely transcribed into cDNA).

1. The primers were designed as follows:

Gm SLT-F 5’-TCTAGAATGGGCGATACTT-3(SEQ ID NO.6)；

Gm SLT-R 5’-GAGCTCTCAAGTCAACATAAGAT-3(SEQ ID NO.7)；

reaction system:

reaction conditions are as follows:

2. the PCR product was purified and recovered using a PCR product purification kit (Shanghai Czeri).

3. Adding A into the purified product

50 μ l reaction:

connecting at 72 ℃ for 30 min;

4. preparation of competent cells of Escherichia coli

1) Inoculating Escherichia coli DH5 alpha, picking single colony in LB medium at 37 ℃ shaking table overnight (about 16 hours);

2) transferring 1ml of overnight culture into 100ml of LB medium, and culturing on a shaker at 37 ℃ for about 2-3 hours (250-300rpm) with vigorous shaking until OD600 is 0.4-0.6;

3) placing 0.1M CaCl2 solution on ice for precooling; the following steps are required to be operated on a clean bench and ice;

4) sucking 1ml of cultured bacterial liquid into a 1.5ml centrifuge tube, and cooling for 10 minutes on ice;

5) centrifuging at 4 deg.C for 5min at 3000 g;

6) discarding the supernatant, adding 100 μ l of pre-cooled 0.1M CaCl2 solution, gently sucking up and down with a pipette gun, stirring well to resuspend the cells, and standing on ice for 20 min;

7) centrifuging at 4 deg.C for 5min at 3000 g;

8) discarding the supernatant, adding 100 μ l of pre-cooled 0.1M CaCl2 solution, and gently sucking up and down with a pipette to resuspend the cells;

9) the cell suspension can be used immediately for transformation experiments or stored at ultra-low temperature (70 ℃) after adding cryoprotectants (15% -40% glycerol).

5. Ligation reaction

TaKaRa pMD18-T simple vector was used, with reference to the instructions.

The molar ratio of the target fragment to the carrier is controlled to be about 6: 1, adding samples and mixing uniformly.

Add equal volume of solution I5. mu.l, mix gently, centrifuge, join at 16 ℃ for several hours.

6. Transformation of

The ligation reaction solution was taken 5. mu.l, and heat shock method was used to transform E.coli DH 5. alpha. competent cells.

1) Taking out the competent cells from-70 ℃, thawing in ice, adding 5 mu l of the ligation reaction solution, lightly mixing, and placing in ice for 30 min;

2) heating in 42 deg.C water bath for 45 s, and rapidly placing in ice for 2 min;

3) adding 890 mul LB liquid culture medium, 180rpm, and culturing in a shaking table for 1 hour;

4) the lower layer of 100. mu.l of the bacterial solution was applied to an LB (Amp/X-gal/IPTG) plate and cultured overnight at 37 ℃ in an inverted state.

7. Colony PCR identification

Picking a white single colony by using a toothpick, putting the single colony into 100 mu l of sterile water, uniformly stirring, and then carrying out colony PCR by using 1 mu l of bacterial liquid as a template. The reaction was carried out in the following 25. mu.l volume under the same conditions as in example (1):

8. extraction of Escherichia coli plasmid

1) A single colony was picked and inoculated into 3-5ml of LB liquid medium containing antibiotics, and shaken overnight at 37 ℃.

2) And (3) putting 1-3ml of bacterial liquid into a clean centrifugal tube, centrifuging for 5min at 12000r/min, discarding the supernatant, and collecting the thalli.

3) Adding 200 μ l of precooled Solution I into the precipitate, shaking and mixing uniformly, and suspending the thallus.

4) Add 200. mu.l of Solution II and mix by quickly and gently inverting the tube count (no more than 5 min).

5) Add 200. mu.l of pre-cooled Solution III, mix gently by reversing the number of centrifuge tubes, and stand on ice for 5 min.

6)12000r/min, and 10min of centrifugation.

7) Carefully pipette the supernatant into another clean centrifuge tube, add 400. mu.l (or equal volume) of Tris saturated phenol: repeatedly shaking chloroform (1: 1) solution gently, mixing, and centrifuging at 12000r/min for 5 min.

8) And (4) sucking the supernatant into another centrifuge tube (avoiding adsorbing protein), adding 400 mu l of chloroform, repeatedly shaking and uniformly mixing, and centrifuging at 12000r/min for 5 min.

9) Carefully pipette the supernatant into another centrifuge tube, add pre-cooled 2 times the volume of absolute ethanol and 1/10 volumes of 3M sodium acetate solution, mix well, and then stand at-20 ℃ for 10 min.

10) Then, the mixture was centrifuged at 12000r/min for 5-10min, and the supernatant was discarded. Washing with 0.5ml of 70% ethanol for 2 times, vacuum drying or air drying, dissolving the precipitate with 50. mu.l of TE + RNase (20. mu.g/ml), and storing at-20 ℃.

9. Plasmid restriction enzyme identification

Using the NEB restriction enzymes BamHI, Sal I, 20. mu.l system

The cleavage was carried out at 37 ℃ for several hours.

10. Sequencing and analysis

Identifying colony PCR and enzyme digestion as positive colony, using single colony LB + Amp to raise for one night, taking a small amount of bacterial liquid to send to company for sequencing, using general primer for sequencing, and obtaining the sequence shown as SEQ ID NO 1; the sequencing results were compared with the GmSLT sequences predicted earlier by the applicant and analyzed using bioinformatics tools such as DNAMAN, NCBI, softberry and Phytozome.

Example 2 Yeast salt tolerance test

The primer is designed to amplify the full length of the salt-tolerant gene ORF, so that the full length of the salt-tolerant gene ORF has double restriction sites which can be connected with a yeast vector pRUL129 to construct a recombinant plasmid. After KOD amplification, ligation was performed to pMD-18T, transformation was performed to DH 5. alpha. by double digestion, overnight extraction of plasmid, recovery and purification of gel, and ligation to pRUL129 vector digested with the same enzyme. The constructed transgenic vector is transformed into saccharomyces cerevisiae W303-1A by a chemical transformation method or an electrical transformation method, and white yeast colonies can be seen after the saccharomyces cerevisiae W303-1A is cultured for 3 days at 28 ℃. And (4) selecting a single colony, and carrying out sequencing verification to be correct after the single colony is cultured for a short time.

1. Competent preparation and transformation of saccharomyces cerevisiae W303-1A by electric transformation method

This step can be performed according to the reference-Qin Yu Jing jin Jian Ling Bao Xiaoming Gao Dong, 1999. Conditions affecting the conversion rate of saccharomyces cerevisiae by electric shock. Shandong university journal (Nature science edition) Vol.34 No. 2.

1) Inoculating a single colony of the yeast strain for transformation into 5ml of YPD medium, and culturing at 30 ℃ overnight until saturation;

2) the evening before transformation, a 2L sterile flask containing 500ml YPD medium was inoculated with an appropriate amount of overnight culture medium and vigorously shaken at 30 ℃ until the cell density reached 1X 10⁸(OD600 of about 1.3-1.5, 1: 10 dilution of about 0.3-0.35);

3) the cultured cells were harvested by centrifugation at 4000g for 5min at 4 ℃ and resuspended in 80ml of sterile water. To increase the sensitivity of the cells to shocks, step 4 is continued. If step 6 is not required;

4) adding 10ml of pH7.5, 10 × TE buffer solution, shaking uniformly, adding 10ml of 1M lithium acetate, rotating and shaking uniformly, and shaking at 30 ℃ and 85rpm for 45 min;

5) 2.5ml of 1mol/L DTT are added while shaking in a rotary manner at 85rpm at 30 ℃ for 15 min;

6) the yeast suspension was washed 3 times, and the cells were pelleted by centrifugation at 4 ℃ with 4000-:

first precipitation: 250ml of ice cold water

And (3) secondary precipitation: 20-30ml ice-cold 1mol/L sorbitol

And (3) third precipitation: 0.5ml of ice-cold 1mol/L sorbitol

The final volume of the bacterial liquid is 1.3-1.5ml, and the density of the bacteria is 1X 10¹⁰。

7) Before electrotransformation, 40. mu.l of yeast cells and 100ng or less of DNA to be transformed (volume less than 5. mu.l) are added into a sterile ice-cold microcentrifuge tube and mixed well. The cells were transferred to an ice-cold electric transfer bath, followed by the operation described in the Bio-Rad electroporator.

8) Adding 1ml of precooled 1mol/L sorbitol into the electric shock tank after the pulse, and slightly blowing, sucking and uniformly mixing;

9) gradient-coated on sorbitol selective medium plate, and cultured at 30 deg.C for 3-6 days until colonies appear on the plate.

2. Extraction of total DNA and plasmid of Saccharomyces cerevisiae W303-1A

1) Centrifuging 1-2ml of yeast liquid cultured for 30h at 12000rpm for 1 min;

2) washing with STE for 2 times;

3) resuspend with 100. mu.l TE buffer, add 50. mu.l glass beads (Sigma Co.), add 100. mu.l phenol chloroform, shake vigorously for 1 h;

4) centrifuging at 12000rpm for 10min at 4 deg.C;

5) taking the supernatant, adding equal volume of chloroform, and extracting protein and phenol;

6) repeating the step 5 until no protein is left at the boundary of the liquid level;

7) taking the supernatant, adding two times of ice absolute ethyl alcohol, standing at-20 ℃ for 20min, and centrifuging at 12000rpm for 10 min;

8) discarding the supernatant, washing once with 70% ethanol, and naturally drying;

9) dissolving in 50-100 μ l TE, adding RNase A (final concentration of 20 μ g/ml), reacting at 37 deg.C for 30min, and storing at-20 deg.C;

10) and (3) PCR detection, wherein the amount of the template is recommended to be 1-2 mul/25 mul of a reaction system because the cracking efficiency is low and the plasmid content is not high.

3. Experiment for influence of overexpression GmSLT gene on growth capacity of yeast under salt stress

The influence of the GmSLT gene on the growth capacity under Yeast salt stress is detected by adopting a spot test (Yeast drop test assay) and a growth curve method (can be referred to in the literature, Chenhongyu, Yeyankee, Zhuyi, Zhengsuiping, forest shadow, 2008. comparison of Yeast tolerance evaluation methods, food and fermentation industries, 2008,34(12): 51-57).

The dropping method is to drop the yeast with the same concentration of wild type and GmSLT on the yeast basic culture medium added with 0,300mM, 500mM NaCl, culture for 2-3 days at 28 ℃, observe the growth condition of the yeast, and take pictures for recording.

The growth curve method comprises the following steps: yeast was inoculated into minimal medium, cultured overnight at 28 ℃ with OD600 adjusted to 0.5, and 100ml of fresh rich medium liquid YPD (containing 300mM NaCl) was inoculated in suspension in 1ml of medium to give an initial OD600 of 0.005, 28 ℃,150rpm, and cultured for 3 days with OD values measured at 6-hour intervals.

As can be seen from FIG. 2, both W303-1A and W303-1A transformed into the empty pRUL129 plasmid grew slowly when the NaCl concentration in the medium reached 300mM, but the W303-1A yeast overexpressing GmSLT still grew when the NaCl concentration in the medium reached 500 mM.

As can be seen from FIG. 3, the growth rate of the yeast W303-1A overexpressing GmSLT was significantly higher than that of the yeast W303-1A overexpressing GmSLT and that of the yeast W303-1A transformed with the empty plasmid pRUL129, starting from 30 hours, when the NaCl concentration in the medium reached 300 mM. By 72h, yeast with the GmSLT transgene had reached 8.85 OD600, whereas the control with the empty plasmid reached only 4.05 and 3.02 for wild type W303-1A.

The experiment proves that the salt stress adaptability of the yeast can be obviously improved by over-expressing the GmSLT gene. Proves that the GmSLT gene function is closely related to salt tolerance.

4. Correlation of each domain of GmSLT and salt-tolerant function

1)Over-Lapping PCR：

In order to verify the function of each domain of GmSLT, deletion of ABS, TM303, TH178 and ZBS domains was performed by Over-mapping PCR, as shown in FIG. 1. According to this method, primers P2 and P3 and common primers P1 and P4 were designed, and 2 cycles of 20-cycle PCR were carried out.

The designed primer sequences are as follows:

GmSLTΔABS-P2 5‘-CTCATGCAA-CCAAGCACCCCAAATG-3‘(SEQ ID NO.8)

GmSLTΔABS-P3 5‘-GGGGTGCTTGG-TTGCATGAGAAATCTAAG-3‘(SEQ ID NO.9)

GmSLTΔTM-P2 5‘-GATTTCTCATG-TTGGTGCACTTCCTTCCAG-3‘(SEQ ID NO.10)

GmSLTΔTM-P3 5‘-GTGCACCAA-CATGAGAAATCTAAGACC-3‘(SEQ ID NO.11)

GmSLTM303-F 5‘-GGATCCATTGATCTATCTCCTGT-3‘(SEQ ID NO.12)

GmSLTM303-R 5‘-GTCGAC-TCAAGTCAACATAAGATCA-3‘(SEQ ID NO.13)

GmSLTH178F-F 5‘-ACATGAACGGGCTCTCTCGCCAAGG-3‘(SEQ ID NO.14)

GmSLTH178F-R 5‘-CCTTGGCGAGAGAGCCCGTTCATGT-3‘(SEQ ID NO.15)

GmSLTΔZBS-P2 5‘-TCTCGGCACT-CCCGTTCATGTAACTCCTC-3‘(SEQ ID NO.16)

GmSLTΔZBS-P3 5‘-CATGAACGGG-AGTGCCGAGAAGGGTTTTG-3‘(SEQ ID NO.17)

P1 5‘-GGATCC-ATGGGCGATACTTCTC-3‘(SEQ ID NO.18)

P4 5‘-GTCGAC-TCAAGTCAACATAAGATCA-3‘(SEQ ID NO.19)

2) construction of Yeast transformation vector recombinant pRUL129

The mutant gene with BamHI and SalI restriction sites verified by sequencing was linked to yeast vector pRUL129 with the same restriction sites, and Saccharomyces cerevisiae was transformed by electrotransformation and cultured on a selection plate containing 1M sorbitol at 28 ℃ for 3 days. A single colony is picked for PCR verification, and the four mutant genes are proved to be transferred into the yeast.

3) GmSLT structural domain and salt tolerance function test

Salt tolerance tests were performed on various transformed yeasts by the method of the spot test (Yeast drop test assay). The results are shown in FIG. 4. It can be observed from FIG. 4 that the yeast overexpressing the complete GmSLT gene or lacking ZBS grew significantly faster than other yeasts when the NaCl concentration in the medium reached 300mM, 500 mM. The experimental result shows that once domains such as ABS, TM and the like are deleted in each domain, the GmSLT gene function is almost completely lost, and the two domains are essential to the salt-resistant function of the GmSLT. In contrast, the functional effect of the GmSLT gene is small after the ZBS structure is deleted.

Example 3 salt tolerance test of GmSLT transgenic rice

1. Construction of plant transformation vectors

Using the modified binary Vector pCAMBIA1301 (reference is old and popular, leaf swallow sharp, Zhuyi, Zheng ear flat, forest shadow, 2008. comparison of yeast tolerance evaluation method. food and fermentation industry, 2008,34(12):51-57) as a plant expression Vector (35S promoter added before multiple cloning site), see example 2, the gene GmSLT was double-digested from the Vector pMD-18T Simple Vector using two restriction sites of BamH I and Sal I, recovered and purified, and then cloned between the multiple cloning sites BamH I and Sal I of pCAMBIA1301, which is called 35S: : GmSLT.

2. Preparation of Agrobacterium

1) And (3) mixing 35S: : the GmSLT is used for transforming agrobacterium LBR4404 (the reference is-Heynun spring, Gaubida, 2002. the construction of a plasmid pBG1121 containing a tobacco chitinase gene and the transformation of rice, Nature science edition, Proc. of Hunan agriculture university, 2002,28(2):93-96), then colony PCR identification is carried out to verify the correctness, and a positive colony is frozen and stored at-70 ℃ with a protective agent. 2) Before transformation, the preserved Agrobacterium is taken out from a refrigerator at-70 ℃, inoculated on a YEP solid culture medium containing corresponding antibiotics, and cultured in an incubator at 28 ℃ for 2-3 d.

3) A single colony of Agrobacterium was picked up and inoculated into 3mL of sterile YEP liquid medium (containing 25mg/L rifampicin +25mg/L streptomycin +50mg/L kanamycin), and cultured at 28 ℃ and 200rpm for about 20 hours.

4) Inoculating the obtained bacterial liquid into 200mL YEP liquid culture medium containing the same antibiotic, culturing at 28 deg.C and 200rpm for about 20h until the concentration of Agrobacterium reaches OD₆₀₀About 0.5.

5) Centrifuging at 4 deg.C and 3000rpm for 15min, discarding supernatant, and resuspending with transformation solution for use.

3. Obtaining of GmSLT gene-transferred rice (reference documents: Zhangxumei, Yuan, Xuxiuzhen, Wangzhijie, Li Chunyang and the like, 2000. construction of plant expression plasmid pBin 438-IFN-gamma and efficient transformation to Agrobacterium LBA 4404. A college institute of Sichuan university (Nature science edition), 2000(s1), easy self-reliance, Cao Dunyun, Wang Li Xiang and the like, 2001. research for improving the frequency of agrobacterium-transferred rice, genetics and reports, 2001,28(4):352 and 358).

There are several methods to obtain transgenic rice, in this example, japonica rice Nipponbare is used as material, callus is induced by mature embryo, then the callus is infected by agrobacterium EHA105 containing GmSLT gene, and finally, transgenic plant is obtained by differentiation.

The method comprises the following specific steps:

1) inducing wound healing and subculture

4.4g/L of induction medium-MS mixed powder; 2, 4-D: 4 mg/L; sucrose: 30 g/L;

proline: 2.8 g/L; hydrolyzing casein: 0.3 g/L; plant gel: 3g/L (PH5.8)

4.4g/L subculture medium-MS mixed powder; 2, 4-D: 2 mg/L; Fe-EDTA: 5 ml/L; sucrose: 30 g/L; hydrolyzing casein: 0.3 g/L; plant gel: 3g/L (PH5.8)

Removing shells of mature rice seeds, disinfecting the seeds with a sodium hypochlorite solution, washing the seeds with sterilized deionized water for multiple times, putting the seeds on sterile filter paper for blotting, flatly placing the seeds on an induction culture medium, and culturing the seeds for about one month in the dark at the temperature of 28 ℃;

when the embryogenic callus grows out, the spherical callus is selected and placed on a subculture medium for subculture at 28 ℃ in the dark for about 1 week.

2) Agrobacterium transformation and co-culture, co-culture medium-N6 bulk: 50 ml/L; b5 trace: 10 ml/L; b5 organic: 1 ml/L; inositol: 0.1 g/L; 2, 4-D: 2 mg/L; Fe-EDTA: 5 ml/L; sucrose: 30 g/L; glucose: 10 g/L; plant gel: 3 g/L; sterilizing, adding AS at 150 μ M (pH 5.2); see example 3 vector 35S: : GmSLT transformed Agrobacterium EHA105 and tested for positive colonies. Activating and culturing positive Agrobacterium with YEP plate containing antibiotic at 28 deg.C for 2d, scraping off the positive Agrobacterium, resuspending in AAM culture solution containing appropriate amount of bacteria, culturing at 28 deg.C for 2 hr under vigorous shaking on shaking table, and adjusting bacterial liquid OD₆₀₀The value is about 0.15. Immediately afterwards, the subcultured callus was soaked in Agrobacterium solution for about 25 min. Then, the culture was blotted dry with sterile filter paper, and the callus was transferred to a co-culture medium containing a piece of sterile filter paper and co-cultured in the dark at 20 ℃ for 3 days.

3) Selection and differentiation culture

Selection medium-N6 bulk: 50 ml/L; b5 trace: 10 ml/L; b5 organic: 1 ml/L; 2, 4-D: 2 mg/L; Fe-EDTA: 5 ml/L; sucrose: 30 g/L; proline: 0.5 g/L; hydrolyzing casein: 0.3 g/L; inositol: 0.1 g/L; phytagel: 3g/L, adding the cefuroxime after sterilization: 500 mg/L; add hygromycin 50mg/L (pH5.8) Pre-alimentation Medium-N6 bulk: 50 ml/L; b5 trace: 10 ml/L; b5 organic: 1 ml/L; NAA: 1 mg/L; Fe-EDTA: 5 ml/L; sucrose: 30 g/L; proline: 0.5 g/L; hydrolyzing casein: 0.3 g/L; inositol: 0.1 g/L; plant gel: 3g/L, adding the cefuroxime after sterilization: 250 mg/L; ABA: 5 mg/L; 6-BA: 2.5 mg/L; adding hygromycin 50 mg/L; (PH5.8)

Differentiation medium-N6 bulk: 50 ml/L; b5 trace: 10 ml/L; b5 organic: 1 ml/L; NAA: 0.5 mg/L; Fe-EDTA: 5 ml/L; 6-BA: 4 mg/L; sucrose: 30 g/L; hydrolyzing casein: 0.3 g/L; inositol: 0.1 g/L; plant gel: 4.6 g/L. (PH5.8)

After co-culture, transferring the callus to a triangular flask, adding sterile water to wash the callus for 3 times, then washing the callus with sterile water added with 0.5g/L of cefuroxime for many times until the callus is clear, pouring out the sterile water, then adding sterile water (containing 0.5g/L of cefuroxime) and placing the callus on a shaking table to shake for 2 hours at 28 ℃ and 150 turns, if the liquid is turbid, washing the callus with 0.5g/L of cefuroxime sterile water until the liquid is clear, then transferring the callus to a filter paper for sterility, sucking off the excess water, blowing the callus on an ultra-clean bench for about 1 hour, then transferring the callus to a selective culture medium (the larger callus can be selected to be dispersed on the culture medium) and culturing the callus for two weeks at 28 ℃ in the dark.

Then selecting the resistant callus growing from the original callus, transferring the resistant callus to a pre-differentiation culture medium, and performing pre-differentiation culture for 1 week at the dark temperature of 28 ℃. Then, the callus was transferred to a differentiation medium, and differentiation culture was performed at 28 ℃: culturing for 3 days in the dark and then culturing for more than two weeks under continuous illumination and photoperiod of 16h day/8 h night, so that the calluses can differentiate into seedlings. Transferring the differentiated seedlings to a triangular flask containing an MS culture medium for culturing and growing into plants (with developed root systems), and finally transplanting the plants to nutrient soil for culturing.

4) Identification of Rice Positive plants

a. General PCR detection of To-generation seedlings of GmSLT rice

A small amount of rice leaves are cut off by an SDS simple extraction method, and DNA is extracted by grinding with liquid nitrogen. Then using this as template, using GmSLT detection primer amplification, PCR amplification was performed, and using original parent rice (WT) as control, see FIG. 5. The primers are as follows:

GmSTL-d-F：5’-CTTCACATGAGGAACTGG-3’(SEQ ID NO.20)

GmSTL-d-R：5’-CTCAGTCTCATAGCCTTG-3’(SEQ ID NO.21)

from the electrophorogram (fig. 5), it can be seen that the same fragments as those amplified by PCR using a plasmid containing the GmSTL gene as a positive control were amplified regardless of the genomic DNA or cDNA of the rice seedlings transformed with GmSTL as a template, demonstrating that the GmSTL gene was successfully introduced into the transgenic seedlings.

5) Gene expression level analysis (Real time PCR) of To generation partial strain of GmSLT rice

a) Total RNA extraction of seedlings was performed by using a plant RNA extraction kit of Beijing kang, a century company, and an operation reference instruction. And (3) treatment of related articles: soaking the blue rubbing stick with 0.1% DEPC solution overnight, sterilizing at 121 deg.C for 30min, pouring out DEPC solution, and oven drying at 80 deg.C. And preparing 75% ethanol by adding DEPC water and absolute ethanol.

b) Reverse transcription into cDNA. Total RNA was extracted from the leaves, the volume of RNA eluted was 50. mu.l, and after checking the purity and integrity of RNA, reverse transcription was performed. Then, cDNA was synthesized using a reverse transcription kit according to the instructions.

Then, RT-PCR detection was performed using the prepared cDNA as a template, and positive plant detection was performed using the primers used in the above general detection, and the original parent rice (WT) was used as a control.

Selecting rice plants which are detected as positive to prepare a cDNA template, and respectively designing real-time fluorescent quantitative PCR primers of ricetub and GmSLT according to related principles as follows:

Rice tub-F：5’-GGCAAGATGAGCACCAAGGA-3’(SEQ ID NO.22)

Rice tub-R：5’-AAGCCACCGCAATACACCAC-3’(SEQ ID NO.23)

GmSLT-F 5’-AGAATCACCACCCATCCA-3’(SEQ ID NO.24)

GmSLT-R 5’-GTGCCAAGACCAACATCC-3’；(SEQ ID NO.25)

a real-time fluorescent quantitative pcr reaction system (SYBR Green staining method) and a 20-microliter system, which comprises the following specific components:

gently mixing and then centrifuging briefly

Reaction conditions are as follows:

Setp1

1×95℃ 5min

the program finally uses the melting curve (melting curve) reaction to analyze the specificity of the PCR product.

Real-time fluorescent quantitative PCR was performed in triplicate for each sample, using rice housekeeping gene tub as internal reference, using 2^-ΔΔCTThe method calculates the relative expression level of the target gene.

As can be seen from FIG. 6, the expression of GmSLT was not detected in the wild-type parent seedlings, while the expression of GmSLT was detected in all of transgenic seedlings No. 1, 3 and 4, with the highest expression level in seedling No. 1.

4. Salt tolerance test of transgenic rice

Materials: wild type WT seedlings of Nipponbare rice and seedlings of To generation 1# lines of GmSLT rice;

the treatment method comprises the following steps: respectively irrigating MS and NaCl solutions with NaCl concentration of 1M and 1.50M, irrigating 30ml once per pot, and counting the salt tolerance of the rice after 9 days.

As can be seen from the following table and FIG. 7, after 9 days of treatment of the 1# plantlet of the parental Nipponbare and the GmSLT with 1M NaCl, the wild Nipponbare showed significant withering phenomenon, while the plantlet of the GmSLT gene showed no significant salt damage. When treated with 1.5M NaCl for 9 days, the wild type Japanese has died basically, while the seedlings transformed with the GmSLT gene have withered leaves, but the seedlings still survive. Therefore, the conclusion can be drawn that the salt tolerance of the rice can be obviously improved by over-expressing the GmSLT gene.

Rice seedling salt stress treatment result of GmSLT gene transfer

Salt concentration	Plant, its production method and use	Plant performance after treatment
			1M	WT Rice	The leaf withered and heavy
1M	Transgenic GmSLT rice	Slight leaf blight
			1.5M	WT Rice	Part of the plants withers and part of the plants wither seriously
1.5M	Transgenic GmSLT rice	Plant survived and leaf withered

In conclusion, the salt-tolerant gene is obtained by bioinformatics and gene space-time expression analysis and is named as GmSLT; the salt tolerance of related organisms can be obviously improved by transforming the gene into various organisms such as yeast, rice and the like. The gene of the invention can be used for improving industrial strains, such as yeast, and crops, such as: the salt tolerance of rice can be obviously improved. Has the application prospect and value of reducing the culture requirements of industrial production strains such as yeast and the like, reducing the production cost, improving the yield of target compounds, expanding the crop planting area, improving the yield and the like.

The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.

Claims

1. An application of nucleic acid for coding GmSLT protein in enhancing the salt tolerance of plants, wherein the amino acid sequence of the GmSLT protein is shown as SEQ ID NO. 2.

2. The use of claim 1, wherein the sequence of the salt-tolerant domain of the GmSLT protein consists of the domain ABS sequence shown as SEQ ID No.3 and the TM sequence shown as SEQ ID No.4, or of the domain ABS sequence shown as SEQ ID No.3, the TM sequence shown as SEQ ID No.4 and the ZBS sequence shown as SEQ ID No. 5.

3. A method for improving the salt tolerance of plants is characterized by comprising the following steps: introducing nucleic acid for coding GmSLT protein into plants, and culturing to obtain transgenic plants with salt tolerance; the nucleic acid sequence of the GmSLT protein is shown as SEQ ID NO. 1.

4. The method of claim 3, wherein the plant is a monocot, a dicot, or a gymnosperm.

5. The method of claim 4, wherein the plant is a crop plant, a floral plant, or a forestry plant.

6. The method of claim 4, wherein the plant is soybean, rice, Arabidopsis, wheat, corn, cotton, canola, sorghum, or potato.

7. A method for growing salt-tolerant plants, comprising the steps of: crossing the transgenic plant obtained by the method of claim 3 with a target plant to obtain a salt-tolerant plant and a filial generation.