Wheat salt tolerance related gene TaOTS1-5A, protein coded by same and application of wheat salt tolerance related gene TaOTS1-5A

Abstract

The invention belongs to the technical field of plant genetic engineering, and provides a wheat salt tolerance related gene TaOTS1-5A, a protein coded by the wheat salt tolerance related gene TaOTS1-5A and application of the wheat salt tolerance related gene TaOTS1-5A to regulation and resistance of salt stress. According to the invention, a wheat salt tolerance related gene TaOTS1-5A is connected to a modified pCAMBIA3301 vector, such that a recombinant vector modified pCAMBIA3301-TaOTS1-5A is obtained; the recombinant vector is transformed by agrobacterium and then introduced into a receptor plant to obtain a TaOTS1-5A transgenic plant, so that overexpression of the TaOTS1-5A gene is realized. The gene TaOTS1-5A capable of resisting salt stress is obtained through cloning for the first time and is transferred into spring wheat Fielder through an agrobacterium tumefaciens-mediated method, and research finds that compared with a wild type, the salt tolerance of the constructed transgenic plant is remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and particularly relates to a wheat salt tolerance-related gene TaOTS1-5A, the protein encoded thereby, and their applications in resisting salt stress. Background Art

[0002] Salt stress is one of the main adverse factors restricting the growth, development and yield formation of crops. Wheat is a major crop mainly planted in arid / semi-arid and saline-alkali areas around the world, and its yield is often affected by soil salinization. In order to maintain normal growth and development in adverse environments such as salt stress, it is necessary to appropriately change its own cell and physiological states to perceive and respond to various interferences or stresses. Therefore, exploring and utilizing salt tolerance-related genes and analyzing their action mechanisms are of great significance for cultivating excellent new wheat varieties and promoting high and stable wheat yields, and are also an urgent need to promote "adapting planting to the land", promoting the transformation and management of saline-alkali land, and ensuring national food security and ecological security.

[0003] TaOTS1-5A is a SUMO protease. Protein ubiquitin-like modification (small ubiquitin-related modifier, SUMOylation) is a post-translational modification of proteins mediated by SUMO proteins, also known as ubiquitin-like or small ubiquitination, and plays an important role in plant growth and development and stress responses. In Arabidopsis thaliana, AtSIZ1 can regulate key transcription factors in the ABA signaling pathway through SUMOylation modification, thereby affecting seed germination and seedling growth. Previous studies have found that OTS1 can also affect signal transduction in plants by promoting or inhibiting the SUMO levels of related proteins. In rice, OsOTS-RNAi rice plants accumulate high levels of SUMO-conjugated proteins during salt stress and are highly sensitive to salt; transgenic rice plants overexpressing OsOTS1 have higher salt tolerance and simultaneously reduce the level of SUMOylated proteins.

[0004] In summary, SUMO proteases play an important role in plant stress resistance. OTS1 has been proven to be a positive regulator of salt tolerance in rice, but there is little research on it in wheat, especially in the research related to resisting salt stress, no reports have been seen. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a wheat salt tolerance-related gene TaOTS1-5A, the protein encoded thereby, and their applications in regulating the resistance to salt stress.

[0006] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions.

[0007] The present invention provides a wheat salt tolerance-related gene TaOTS1-5A, and the nucleotide sequence of the wheat salt tolerance-related gene TaOTS1-5A is shown in SEQ ID NO.1:

[0008]

[0009] The present invention also provides a protein encoded by the wheat salt tolerance-related gene TaOTS1-5A. The amino acid sequence of the protein encoded by the wheat salt tolerance-related gene TaOTS1-5A is shown in SEQ ID NO.2:

[0010] MTMGASSSNRIHIDWVQMFSPASPGRDDDVCFDSPPSAAGKGRAKGNASARSSPGEGFVSPPPAPAKGKGRRDAEAEGEFSGRSDDGPRRDIADLSGEELRLQITCVRSTHPAFGGLIEQEKREGRKRLLRLLEEEARRRGMGTNSAKDGGASYRDPRSDAYLLDVDDSEAERAHRYHLKLSPIRSTKKNYGGLGVVARRSLKQPGRMRPIPEDKMYSSKTSPSTLSGHKQRVRAVDPKEHDREKRRQIQSNFFSNPANRWNVQHGDSSVSYSRKVNDVVLVDDEDAQSDEPVDCRVPEEWNYSKIYYPSRDDPEAVELTSSDIKCLDPGVYLSSPVINYYIQYIKRDKFQREAARNNFHMFHTYFYSKLQEALFGKGEFVKLRRWWKGVNIFQRGYIILPIHGTAHWSLVIICIPAKESNSGPIVLHLDSLGMHPTDDIYRTVRRFLEEEWKHLRKNPPSDISISDTIWEDLPRNIHKENVEVPGQNNAYDCGIFMLYYIQRFIIEAPENFTRDRLVMFSRSWFRSEEASNLRNKIRKLLLKEFESARVDDVMSEAATADGSDDDCFMKEGESEAPTADGSHEDCVMMEGESEAVIPEAATADGSDDVKTGAATSDGSDEILWKGKSEAVASRDSDLMVVGGGDTFGGTPWSTRKSDGRNRVCVLSEEATLPGSTVKDDEYSMKSDPDSSESEEVVEFLPSDNDNDNDNEEVMHRGTRPDLFYCDDSCDSEAEEVTGAWKRRSRTMKRPDRAGDVQIIEDRKPRARLELCRMT.

[0011] The present invention also provides a recombinant vector overexpressing the wheat salt tolerance-related gene TaOTS1-5A. The recombinant vector includes an initial expression vector and the wheat salt tolerance-related gene TaOTS1-5A. Among them, the nucleotide sequence of the wheat salt tolerance-related gene TaOTS1-5A is shown in SEQ ID NO.1.

[0012] Furthermore, the initial expression vector is the modified pCAMBIA3301 vector (the insertion position of the target gene is between the Sac I and Spe I restriction enzyme sites).

[0013] The present invention also provides a recombinant bacterium overexpressing the wheat salt tolerance-related gene TaOTS1-5A, which includes the above-mentioned recombinant vector. Furthermore, the recombinant bacterium uses Agrobacterium as the original bacterium, and the Agrobacterium is preferably Agrobacterium tumefaciens GV3101.

[0014] The present invention also provides a method for overexpressing the wheat salt tolerance-related gene TaOTS1-5A, which includes the following steps.

[0015] (1) Connect the wheat salt tolerance-related gene TaOTS1-5A to the modified pCAMBIA3301 vector to obtain the recombinant vector modified pCAMBIA3301-TaOTS1-5A.

[0016] The specific steps are as follows:

[0017] Linearize the modified pCAMBIA3301 vector by restriction enzyme digestion: Add 1 μg of the modified pCAMBIA3301 vector, 1 μL of Sac I enzyme, 1 μL of Spe I enzyme, and 3 μL of 10×QuickCut Buffer to a PCR tube, and make up the volume to 30 μL with ddH2O. After preparing the system, incubate at 37 °C for 30 min for restriction enzyme digestion and at 80 °C for 10 min for heat inactivation of the enzyme; obtain the linearized modified pCAMBIA3301 vector.

[0018] Ligate the recombinant vector: Add 2 μL of the linearized modified pCAMBIA3301 vector, 1 μL of the gel recovery product of the target gene TaOTS1-5A, and 3 μL of 2×MonCloneTM Hi-Fusion Cloning Mix to a PCR tube. After preparing the system, incubate at 50 °C for 30 min for the reaction; obtain the recombinant ligation product.

[0019] Recombinant vector transformation: ① Add the reaction solution of the recombinant product to 100 μL of competent cells, slowly pipette and mix well, and place on ice for 30 min; ② Heat shock at 42 °C for 45 - 60 sec, and place on ice bath for 2 min; ③ Add 500 μL of LB medium, and culture with shaking at 37 °C for 40 - 60 min (200 rpm); ④ Spread the bacterial solution evenly on the plate containing the corresponding antibiotic, and incubate upside down at 37 °C overnight.

[0020] Identification of positive clones (colony PCR method): Pick a single colony and mix it in 10 μL of ddH2O. Take 1 μL of the lysate as the template, and use the universal primers M13F: 5’-GACGCACAATCCCACTATCC-3’ (SEQ ID NO.15) and M13R: 5’-GTGCAGATGAACTTCAGGGTCAG-3’ (SEQ ID NO.16) for colony PCR identification to obtain a single colony containing the recombinant vector. Send the single colony to Qingke Biotechnology Co., Ltd. for Sanger sequencing verification to obtain the recombinant vector and strain containing the correct target gene TtOTS1 gene sequence.

[0021] Strain propagation and recombinant vector extraction: Inoculate the strain containing the correct recombinant vector into the LB liquid medium containing 40 - 60 μg / mL kanamycin, culture overnight at 37 °C, centrifuge to collect the bacterial cells, and extract the recombinant vector with reference to the Tiangen plasmid miniprep kit.

[0022] (2) After transforming the above recombinant vector by Agrobacterium, introduce it into the recipient plant to obtain TaOTS1-5A transgenic plants, and achieve the overexpression of the wheat salt tolerance-related gene TaOTS1-5A. Among them, Agrobacterium is preferably Agrobacterium tumefaciens GV3101.

[0023] The preparation method of Agrobacterium tumefaciens GV3101 competent cells is: culture the Agrobacterium tumefaciens GV3101 strain on an LB plate, pick a single colony and inoculate it into the LB liquid medium containing 40 - 60 μg / mL rifampicin for two cultures, collect the bacterial cells, and then suspend the bacterial cells in the CaCl2 solution and the mixed solution of CaCl2 and glycerol in sequence for storage for later use.

[0024] Among them, the concentration of rifampicin in the LB liquid medium is 45 - 55 μg / mL; the dosage of the LB liquid medium containing 40 - 60 μg / mL of rifampicin for the first culture is 3 - 7 mL, preferably 4 - 6 mL; the dosage of the LB liquid medium containing 40 - 60 μg / mL of rifampicin for the second culture is 40 - 60 mL, preferably 45 - 55 mL; the culture temperature is 25 - 32 °C, preferably 26 - 30 °C; the culture rotation speed is 180 - 220 rpm / min, preferably 190 - 210 rpm / min; the culture is carried out until the OD600 value reaches 0.3 - 0.7, and further cultured until the OD600 value reaches 0.4 - 0.6; the concentration of the CaCl2 solution is 0.05 - 0.15 M, preferably 0.08 - 0.12 M; the concentration of CaCl2 in the mixed solution of CaCl2 and glycerol is 0.05 - 0.15 M, preferably 0.08 - 0.12 M; the mass concentration of glycerol is 20 - 40%, preferably 25 - 35%; the volume ratio of CaCl2 to glycerol in the mixed solution of CaCl2 and glycerol is 1:1; the suspension temperature is 2 - 7 °C, preferably 3 - 6 °C; the suspension rotation speed is 3800 - 4200 rpm / min, preferably 3900 - 4100 rpm / min; the suspension time is 8 - 12 min, preferably 9 - 11 min.

[0025] The Agrobacterium tumefaciens GV3101 competent cells and the recombinant vector pUbi-35S-TtOTS1-GFP are mixed and quickly frozen and heat shocked, and then cultured successively with antibiotic-free liquid LB medium and LB plate medium containing rifampicin and kanamycin to obtain the Agrobacterium strain containing the recombinant expression vector modified pCAMBIA3301-TaOTS1-5A.

[0026] Among them, the quick-freezing method is liquid nitrogen quick-freezing, and the quick-freezing time is 8 - 12 min, preferably 9 - 11 min; the heat shock method is water bath heat shock, the heat shock temperature is 34 - 40 °C, preferably 35 - 38 °C; the heat shock time is 3 - 7 min, preferably 4 - 6 min; the dosage of the antibiotic-free liquid LB medium is 0.3 - 1 mL, preferably 0.4 - 0.8 mL; the concentration of rifampicin in the LB plate medium containing rifampicin and kanamycin is 40 - 60 μg / mL, preferably 45 - 55 μg / mL; the concentration of kanamycin in the LB plate medium containing rifampicin and kanamycin is 40 - 60 μg / mL, preferably 45 - 55 μg / mL; the culture method is dark culture, the culture temperature is 24 - 32 °C, preferably 25 - 30 °C, and the culture time is 48 - 54 h, preferably 44 - 50 h.

[0027] The Agrobacterium strain containing the recombinant expression vector modified pCAMBIA3301-TaOTS1-5A was sent to the wheat transgenic platform of the State Key Laboratory of Crop Stress Resistance and High-Efficiency Production, Northwest A&F University for subsequent work. The transgenic receptor line was the spring wheat Fielder line. The specific steps were the induction of callus, the infection of wheat young embryos with Agrobacterium, the resistance screening of callus, the differentiation of callus, and the obtaining of plants overexpressing the salt tolerance-related gene TaOTS1-5A of wheat.

[0028] The present invention also provides the application of a salt tolerance-related gene TaOTS1-5A of wheat, a protein encoded by the salt tolerance-related gene TaOTS1-5A of wheat, a recombinant vector overexpressing the salt tolerance-related gene TaOTS1-5A of wheat, or a recombinant bacterium overexpressing the salt tolerance-related gene TaOTS1-5A of wheat in regulating the ability of plants to resist salt stress, that is, overexpressing the salt tolerance-related gene TaOTS1-5A of wheat in plants to improve the salt tolerance of plants.

[0029] Preferably, the above-mentioned plant is wheat.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The present invention utilizes the existing plant genetic engineering technology to clone for the first time the gene TaOTS1-5A that regulates the resistance to salt stress, and transfers this gene into the spring wheat variety Fielder by the method mediated by Agrobacterium tumefaciens. Through comparative test analysis, it is proved that the transgenic plants constructed by the gene of the present invention have significantly improved salt tolerance compared with the wild type, and further confirmed that the gene provided by the present invention plays an important role in regulating the resistance to salt stress and can be used as an important gene resource for cultivating new salt-tolerant crop varieties. Description of the Drawings

[0032] Figure 1 Amplification result of the full-length CDS sequence of TaOTS1-5A gene (M is D5000 marker);

[0033] Figure 2 PCR detection result of Bar gene (M is D2000 marker);

[0034] Figure 3 Detection result of the expression level of TaOTS1-5A transgenic wheat;

[0035] Figure 4 Expression levels of TaOTS1-5A gene in different tissues (roots, stems, flag leaves, awns, and grains) at the heading stage;

[0036] Figure 5Expression levels of the proline synthesis-related gene TaP5CS1 in TaOTS1-5A transgenic wheat lines under different treatments;

[0037] Figures 6 to 13 Phenotypic results of salt tolerance identification of TaOTS1-5A transgenic wheat seedlings at the seedling stage; among them, Figure 6 Phenotypes of wild-type and transgenic lines under normal cultivation, Figure 7 Phenotypes of wild-type and transgenic lines treated with 200 mM NaCl for 10 days, Figures 8 to 13 Salt tolerance indices of leaf length, root length, leaf fresh weight, root fresh weight, leaf dry weight, and root dry weight of wild-type and transgenic lines, respectively. Specific implementation mode

[0038] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments.

[0039] Experimental materials: The wheat variety Chinese Spring (Chinese Spring, English name Chinese Spring) of common wheat (Triticum aestivum L.) is a very important local wheat variety. Chinese Spring is widely used in wheat genetics research and can be obtained by the public from the applicant's team (Wheat Genomics and Molecular Breeding Team, College of Agronomy, Northwest A&F University).

[0040] Example 1

[0041] Full-length cloning of the TaOTS1-5A gene CDS.

[0042] 1. Take Chinese Spring wheat seeds of uniform size, after disinfection and sterilization, sow them on filter paper, and treat them at 4 °C for 3 days until they show white tips. Then transfer the germinated seeds to nutrient soil and culture them at 22 °C for two weeks to obtain wheat seedlings.

[0043] 2. Take Chinese Spring wheat seedlings, quickly freeze them in liquid nitrogen, and store them at -80 °C for later use. Extract the total RNA of wheat leaves using the Trizol method (TianGen), and then reverse transcribe it to obtain cDNA using the PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara).

[0044] 3. Using the wheat cDNA as a template, perform PCR amplification with the primer pair composed of 5’-ATGACGATGGGGGCGTCGTCGTC-3’ (SEQ ID NO.3) and 5’-CCTGGAAATGGGTGGATGAGC-3’ (SEQ ID NO.4) to obtain the PCR amplification product of the full-length CDS of TaOTS1-5A. The PCR product was detected by 1.0% agarose gel electrophoresis (as Figure 1 shown).

[0045] The PCR reaction program was as follows: pre-denaturation at 95°C for 30 min; denaturation at 95°C for 30 sec, annealing at 57°C for 30 sec, extension at 72°C for 5 min, with 35 cycles; extension at 72°C for 10 min.

[0046] 4. Ligate the PCR amplification product to a cloning vector, transform it into Escherichia coli, and pick monoclonal colonies for sequencing.

[0047] Experimental results: The sequencing results showed that the nucleotide sequence CDS of the PCR amplification product was as shown in SEQ ID NO.1. The gene shown in SEQ ID NO.1 was named TaOTS1-5A gene. The TaOTS1-5A gene encodes TaOTS1-5A protein, and the amino acid sequence of TaOTS1-5A protein was as shown in SEQ ID NO.2.

[0048] Example 2

[0049] Establishment of TaOTS1-5A transgenic wheat plants.

[0050] Verify the function of TaOTS1-5A gene using transgenic wheat, and the specific process is as follows.

[0051] 1. Obtaining TaOTS1-5A transgenic wheat

[0052] Using the correctly sequenced recombinant cloning plasmid obtained in Example 1 as a template, 5’-AGAGGGGGATTAACTAGTATGACGATGGGGGCGTCGTCGTCGAATAGGAT-3’ (SEQ ID NO.5) and 5’-ACCACCGCTACCGAGCTCCGTCATGCGGCAGAGTTCCAATCTAGCTCT-3’ (SEQ ID NO.6) as primers for PCR product amplification, and integrate the modified pCAMBIA3301-TaOTS1-5A gene into the wheat genome by Agrobacterium (GV3101)-mediated genetic transformation method. This process was completed by the wheat transgenic platform of the State Key Laboratory of Crop Stress Biology in Arid Areas, Northwest A&F University. The transgenic receptor line was the spring wheat Fielder line. The specific steps were induction of callus, Agrobacterium infection of wheat immature embryos, resistance screening of callus, differentiation of callus, and acquisition of transgenic plants.

[0053] 2. Identification of positive transgenic wheat plants

[0054] To reduce the probability of false positive results, PCR was performed to identify positive transgenic wheat lines and real-time fluorescence quantitative PCR was used to detect the expression levels of transgenic lines.

[0055] Extract the DNA of transgenic wheat leaves, and screen for transgenic positive plants by detecting whether the Bar gene exists in the genomic DNA samples of regenerated plants through PCR. The primer pair for detecting the expression level of the Bar gene is 5’-CGTCAACCACTACATCGAGACAAG-3’ (SEQ ID NO.7) and 5’-GGTACCGGCAGGCTGAAGTC-3’ (SEQ ID NO.8).

[0056] The PCR reaction procedure is as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 sec, annealing at 57°C for 30 sec, extension at 72°C for 30 sec, for 35 cycles; extension at 72°C for 10 min.

[0057] The PCR products were detected by 1.0% agarose gel electrophoresis (as Figure 2 shown).

[0058] Extract the RNA of wild-type and transgenic wheat lines leaves, and reverse transcribe to obtain the cDNA of wild-type and transgenic wheat lines. Using the cDNA of wild-type and transgenic wheat lines as templates, the relative expression levels of TaOTS1-5A gene were detected by real-time fluorescence quantitative PCR (using TaEF gene as the internal reference gene). The primer pair for detecting the expression level of TaOTS1-5A gene is 5’-CTCTGATGATGTTAAGACAGGGGC-3’ (SEQ ID NO.9) and 5’-CGAATATTCATCGTCCTTCACCGTG-3’ (SEQ ID NO.10). The primer pair for the selected internal reference TaEF gene is 5’-GCTGACTGTGCTGTTCTCATCATC-3’ (SEQ ID NO.11) and 5’-GCGCCTTTGAGTACTTGGGAG-3’ (SEQ ID NO.12), and the results of real-time fluorescence quantitative PCR are as Figure 3 shown.

[0059] Experimental results: As Figure 2 shown, the position of the Bar gene band is correct and bright. As Figure 3 shown, the relative expression levels of TaOTS1-5A gene in the three transgenic wheat lines are all significantly higher than those of the wild type, indicating that TaOTS1-5A has been successfully overexpressed in the transgenic lines.

[0060] Example 3

[0061] 1. Take the roots, stems, flag leaves, awns, and grains of T2 generation TaOTS1-5A transgenic wheat plants at the milk ripening stage, quickly freeze them in liquid nitrogen, and store them at -80 °C for later use. Extract the total RNA of wheat leaves using the Trizol method (TianGen), and then reverse transcribe it into cDNA using the PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara).

[0062] Using the cDNA of each tissue of TaOTS1-5A transgenic wheat as a template, real-time fluorescence quantitative PCR was used to detect the relative expression levels of TaOTS1-5A in each tissue (using the TaEF gene as an internal reference gene). The primer pair for detecting the expression level of the TaOTS1-5A gene was 5’-CTCTGATGATGTTAAGACAGGGGC-3’ (SEQ ID NO.9) and 5’-CGAATATTCATCGTCCTTCACCGTG-3’ (SEQ ID NO.10). The primer pair for the selected internal reference TaEF gene was 5’-GCTGACTGTGCTGTTCTCATCATC-3’ (SEQ ID NO.11) and 5’-GCGCCTTTGAGTACTTGGGAG-3’ (SEQ ID NO.12). The results of real-time fluorescence quantitative PCR are as Figure 4 shown.

[0063] Experimental results: As Figure 4 shown, the expression level of TaOTS1-5A transgenic wheat is the highest in the roots.

[0064] Example 4

[0065] Expression analysis of salt stress-related genes in TaOTS1-5A transgenic wheat.

[0066] 1. Take transgenic wheat seeds of different TaOTS1-5A lines with uniform size, after disinfection and sterilization, sow them on filter paper, treat them at 4 °C for 3 days, and then transfer the germinated seeds to nutrient solution and culture them at 22 °C for two weeks to obtain TaOTS1-5A transgenic wheat seedlings.

[0067] 2. After completing step 1, transfer a part of the TaOTS1-5A transgenic wheat seedlings to nutrient solution containing 200 mM NaCl and treat them for 3, 6, and 9 days; collect the leaves and roots of the treatment group and the control group, quickly freeze them in liquid nitrogen, and store them at -80 °C for later use.

[0068] 3. Total RNA of wheat leaves was extracted using the Trizol method (TianGen), and then cDNA was obtained by reverse transcription using the PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara).

[0069] 4. After completing step 3, using the cDNA of TaOTS1-5A transgenic wheat as a template, real-time fluorescence quantitative PCR was performed to detect the expression analysis of salt stress-related genes in TaOTS1-5A transgenic wheat (using the TaEF gene as an internal reference gene). The primer pair for detecting the proline synthesis-related gene TaP5CS1 was 5’-GATTCTCCGATGGTGCTCGT-3’ (SEQ ID NO.13) and 5’-CCCTTTCCCTCGTAAGAGCC-3’ (SEQ ID NO.14). The primer pair for detecting the expression level of the TaEF gene was 5’-GCTGACTGTGCTGTTCTCATCATC-3’ (SEQ ID NO.11) and 5’-GCGCCTTTGAGTACTTGGGAG-3’ (SEQ ID NO.12).

[0070] Experimental results: The detection results are as Figure 5 shown; the results indicate that under normal growth conditions, there was no significant difference in the transcriptional levels of the TaP5CS1 gene between WT and overexpressing wheat lines. However, after salt treatment, the gene expression level of the salt stress proline synthesis-related gene TaP5CS1 increased in TaOTS1-5A transgenic wheat. Proline is another important osmoprotectant accumulated by plant cells under stress conditions. It can not only stabilize proteins and cell structures but also participate in the stress response of plants as an anti-stress signaling molecule. This example further confirmed that TaOTS1-5A is a positive regulator of wheat salt tolerance, and it is preliminarily speculated that TaOTS1-5A may resist salt stress by regulating osmotic regulators, ultimately making the overexpressing wheat exhibit better salt tolerance.

[0071] Example 5

[0072] Overexpression of TaOTS1-5A improves wheat salt tolerance.

[0073] Salt tolerance phenotype identification at the seedling stage: Take TaOTS1-5A transgenic wheat seeds of uniform size. After disinfection and sterilization, they were sown on filter paper and treated at 4°C for 3 days. Then, the germinated seeds were transferred to nutrient solution and cultured at 22°C for 7 days to obtain TaOTS1-5A transgenic wheat seedlings; then, a part of the TaOTS1-5A transgenic wheat seedlings were transferred to nutrient solution containing 200 mM NaCl and treated with salt stress for 10 days. The phenotypes of the control group and the treatment group were observed and data were statistically analyzed.

[0074] Experimental results: The phenotypic results of the salt tolerance identification of TaOTS1-5A transgenic wheat seedlings are as follows Figures 6 - 13 shown; the results show that under normal cultivation, the transgenic lines and the wild type grow well without obvious distinction; after 10 days of salt stress at the seedling stage, the growth of both the transgenic lines and the wild type is restricted, but the overexpression of TaOTS1-5A alleviates this restriction, with longer root lengths and better growth conditions; the above results indicate that the overexpressed gene TaOTS1-5A can improve the salt tolerance of transgenic plants.

[0075] As can be seen from the above examples, the overexpressed gene TaOTS1-5A of the present invention can significantly improve the salt tolerance of transgenic plants compared to the wild type.

Claims

1. A wheat salt tolerance-related gene TaOTS1-5A, characterized in that, The nucleotide sequence of the wheat salt tolerance-related gene TaOTS1-5A is shown in SEQ ID NO.

1.

2. The protein encoded by the wheat salt tolerance-related gene TaOTS1-5A, characterized in that, The amino acid sequence of the protein encoded by the wheat gene TaOTS1-5A is shown in SEQ ID NO.

2.

3. A recombinant vector overexpressing the wheat salt tolerance-related gene TaOTS1-5A, characterized in that, The recombinant vector includes an initial expression vector and the wheat salt tolerance-related gene TaOTS1-5A, and the nucleotide sequence of the wheat salt tolerance-related gene TaOTS1-5A is shown in SEQ ID NO.

1.

4. The recombinant vector according to claim 3, characterized in that, The initial expression vector is the modified pCAMBIA3301 vector.

5. A recombinant bacterium overexpressing the wheat salt tolerance-related gene TaOTS1-5A, characterized in that, The recombinant bacterium includes the recombinant vector described in claim 3 or 4.

6. A method for overexpressing the wheat salt tolerance-related gene TaOTS1-5A, characterized in that, Comprising the following steps: Connecting the wheat salt tolerance-related gene TaOTS1-5A to the modified pCAMBIA3301 vector to obtain the recombinant vector modified pCAMBIA3301-TaOTS1-5A; After transforming the above recombinant vector through Agrobacterium, introducing it into the recipient plant to obtain TaOTS1-5A transgenic plants, and realizing the overexpression of the wheat salt tolerance-related gene TaOTS1-5A.

7. Use of the wheat salt tolerance-related gene TaOTS1-5A according to claim 1, the protein according to claim 2, the recombinant vector according to claim 3 or 4, or the recombinant bacterium according to claim 5 in regulating the salt stress resistance ability of plants, characterized in that, Overexpressing the wheat salt tolerance-related gene TaOTS1-5A in plants to improve the salt tolerance of plants.

8. The application according to claim 7, wherein The plant is wheat.

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