Application of wheat stress-resistant protein TaFIP37-7B and coding gene thereof in regulation and control of plant stress resistance

Abstract

The invention relates to the technical field of genetic engineering, in particular to application of wheat stress-resistant protein TaFIP37-7B and a coding gene thereof in regulation and control of plant stress resistance. The invention provides a wheat stress-resistant protein TaFIP37-7B and application of a coding gene of the wheat stress-resistant protein TaFIP37-7B in regulation and control of plant stress resistance. A TaFIP37-7B coding gene derived from wheat is introduced into a receptor plant to obtain a transgenic plant with an overexpressed TaFIP37-7B gene, and a test result proves that compared with a control receptor plant, under drought and salt stress conditions, overexpressed arabidopsis thaliana and wheat show higher drought and salt stress tolerance, and the drought and salt stress tolerance is higher than that of the control receptor plant. It is shown that the TaFIP37-7B protein can significantly improve the drought and salt stress resistance of plants.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly to the application of wheat stress-resistant protein TaFIP37-7B and its encoding gene in regulating plant stress resistance. Background Art

[0003] In recent years, RNA modifications represented by m6A methylation modification, as an epigenetic transcriptional regulation method at the post-transcriptional level, the plant biological processes and enzyme systems involved have been gradually analyzed. As a dynamic modification type, m6A modification is dynamically regulated by methyltransferases (writers) and demethylases (erasers), and the methylation sites are recognized by m6A recognition proteins (readers), which are closely related to gene expression regulation and protein translation, thereby playing corresponding biological functions. Existing studies have shown that m6A methyltransferases play important roles in plant growth and development and stress responses: regulating plant growth, fruit development, abiotic stress responses, etc.

[0004] In plants, the functional research of m6A methyltransferases mainly focuses on Arabidopsis thaliana. The research on m6A methyltransferases in crops is still in its infancy. In particular, the regulatory role of m6A methyltransferases in wheat drought resistance and salt tolerance has not been reported. In view of this, based on the wheat m6A methyltransferases identified by whole-genome in the early stage, this study screened the methyltransferase-encoding gene TaFIP37-7B that was significantly induced by drought and salt stress, constructed overexpression and gene-editing materials through Agrobacterium-mediated transformation technology, and performed phenotypic identification on each strain to clarify its function in regulating plant drought resistance and salt tolerance, with a view to enriching wheat stress-resistant gene resources and laying a foundation for cultivating stress-resistant wheat. Summary of the Invention

[0005] The purpose of the present invention is to provide the application of wheat stress-resistant protein TaFIP37-7B and its encoding gene in regulating plant stress resistance.

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

[0007] The present invention provides the application of TaFIP37-7B gene or biological material containing TaFIP37-7B gene in any one of the following:

[0008] a. For regulating the drought resistance of plants;

[0009] b. For regulating the salt stress tolerance of plants;

[0010] c. For breeding drought-resistant plant varieties;

[0011] d. For breeding salt-tolerant plant varieties;

[0012] e. For improving the drought resistance and salt tolerance of plant germplasm resources;

[0013] The nucleotide sequence of the TaFIP37-7B gene is shown in SEQ ID NO.2.

[0014] Preferably, the amino acid sequence of the protein encoded by the TaFIP37-7B gene is shown in SEQ ID NO.1.

[0015] Preferably, the biological material includes any one of the following:

[0016] d1. An expression cassette containing the TaFIP37-7B gene;

[0017] d2. A recombinant vector containing the TaFIP37-7B gene or a recombinant vector containing the expression cassette described in d1;

[0018] d3. A recombinant microorganism containing the expression cassette described in d1, or a recombinant microorganism containing the recombinant vector described in d2;

[0019] d5. A transgenic plant cell line containing the expression cassette described in d1, or a transgenic plant cell line containing the recombinant vector described in d2;

[0020] d6. A transgenic plant tissue or organ containing the expression cassette described in d1, or a transgenic plant tissue or organ containing the recombinant vector described in d2.

[0021] Preferably, the recombinant vector is an overexpression vector.

[0022] Preferably, the application is to improve the drought resistance and salt tolerance of the recipient plant by upregulating the expression, activity or content of the protein encoded by the TaFIP37-7B gene in the recipient plant.

[0023] Preferably, the plant is wheat or Arabidopsis thaliana.

[0024] The present invention provides a method for improving the drought resistance and salt tolerance of plants, which improves the drought resistance and salt tolerance of the recipient plant by upregulating the expression, activity or content of the TaFIP37-7B protein in the recipient plant; the TaFIP37-7B protein is any one of the following proteins:

[0025] a1). A protein encoded by the amino acid shown in SEQ ID No.1;

[0026] a2). A fusion protein containing the protein described in a1).

[0027] Preferably, the up-regulation of the expression, activity or content of TaFIP37-7B protein in the recipient plant is specifically achieved by introducing the TaFIP37-7B gene into the recipient plant; the nucleotide sequence of the TaFIP37-7B gene is shown in SEQ ID NO.2.

[0028] Preferably, the recipient plant is Arabidopsis thaliana or wheat.

[0029] The amino acid sequence of the wheat zinc finger protein TaC3H112-6B of the present invention is shown in SEQ ID NO.1:

[0030] MADPPSPRLDEDDTFGRDFNASPSRNAAPARSGEKRQFGDLDDDEDDVFASKKGKTKVEESAPGAATGMILSLRESLQTCKENLESNQVELEAAKSEIQKWHSAFQNIPAVPAGTNPEPVSVITYLSNLKSSEESLKEQLEKAKKREAAFIVTFAKREQEIAELKSAVRDLKTQLRPPSMQTRRLLLDPAIHEEFTRLKNLAEEKEKKIKELQENVAAVNFTPSSKHGKMLMAKCRTLQEENEEIGAMASEGKIHELGMKIAVLKTQNNELRNQFDGLYKHMDGLTNDVERSNEMVSILQEELEAKDVELARLKEMLSQKEATEDDAVVEEREEAANDMNASSDPQPIKAES

[0031] The nucleotide sequence of the wheat zinc finger protein TaC3H112-6B gene of the present invention is shown in SEQ ID NO.2:

[0032]

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] In the present invention, the TaFIP37-7B encoding gene derived from wheat is introduced into a recipient plant to obtain a transgenic plant overexpressing the TaFIP37-7B gene. The test results prove that, compared with the control recipient plants, under drought and salt stress conditions, the overexpressing Arabidopsis thaliana and wheat show stronger drought and salt stress tolerance, indicating that the TaFIP37-7B protein can significantly improve the ability of plants to resist drought and salt stress. The gene for regulating plant stress resistance of the present invention has important significance and application value for cultivating new stress-resistant plant varieties.

[0035] The present invention discloses the application of a wheat m6A methyltransferase TaFIP37-7B protein and its coding sequence in improving plant drought and salt tolerance. Using existing plant genetic engineering techniques, this gene is transferred into Arabidopsis thaliana and wheat through the method mediated by Agrobacterium tumefaciens to increase the activity or content of the TaFIP37-7B protein in the recipient plants. Through comparative analysis, it is proved that the drought and salt tolerance of the overexpressing plants constructed with the gene of the present invention is significantly improved. It is confirmed that the TaFIP37-7B protein and its coding gene of the present invention have important significance in the research of improving plant drought and salt tolerance, can be used as a gene resource for crop genetic improvement applications, and further obtain drought and salt-tolerant wheat or other crops through genetic transformation methods, having broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the provided drawings.

[0037] Figure 1 It is for the cloning of the TaFIP37-7B gene.

[0038] Figure 2 It is for the expression analysis of TaFIP37-7B transgenic Arabidopsis thaliana. Among them, A is the PCR identification result of transgenic Arabidopsis thaliana, and B is the identification result of the expression level of the T3 generation transgenic Arabidopsis thaliana (Col-0 refers to wild-type Arabidopsis thaliana, and OE-1, OE-2, and OE-3 refer to 3 independent transgenic lines of the TaFIP37-7B gene).

[0039] Figure 3Phenotypic results of drought resistance identification of TaFIP37-7B transgenic Arabidopsis thaliana (A is the drought resistance phenotype at the germination stage, B is the statistical result of the germination rate; C is the main root length phenotype at the seedling stage, D is the statistical result of the main root length; E is the drought resistance phenotype at the adult seedling stage, F is the statistical result of the survival rate).

[0040] Figure 4 Phenotypic results of salt tolerance identification of TaFIP37-7B transgenic Arabidopsis thaliana (A is the salt tolerance phenotype at the germination stage, B is the statistical result of the germination rate; C is the main root length phenotype at the seedling stage, D is the statistical result of the main root length).

[0041] Figure 5 Schematic diagram of the overexpression vector of TaFIP37-7B gene in wheat and the results of transgenic verification (A is the schematic diagram of the overexpression vector structure; B is the PCR identification result of TaFIP37-7B transgenic wheat, where M represents Marker, 1-7 represent 7 different independent lines, "+" is the positive control (expression vector), "-" is the negative control; C is the identification result of the expression level of TaFIP37-7B in T3 generation transgenic wheat, Fielder (WT) refers to the transgenic receptor, OE-1, OE-2, OE-3 are 3 independent transgenic lines).

[0042] Figure 6 Phenotypic results of drought resistance identification of TaFIP37-7B transgenic wheat (A is the seedling morphology before drought treatment; B is the seedling morphology 3 days after rehydration after drought; C is the statistical result of the survival rate 3 days after rehydration after drought stress; D is the identification result of the water loss rate of three-leaf stage seedlings; E is the relative water content of leaves under normal conditions and mild drought stress of seedlings).

[0043] Figure 7 Phenotypic results of salt tolerance identification of TaFIP37-7B transgenic wheat (A is the plant morphology of hydroponic plants under normal conditions; B is the plant morphology after salt stress treatment; C is the statistical result of the biomass of plants under normal conditions and salt stress treatment; D is the calculated salt tolerance coefficient). Detailed implementation mode

[0044] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0045] Example 1 Obtaining of TaFIP37-7B protein and its coding gene

[0046] 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.

[0047] 2. Take 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 into cDNA using the PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara).

[0048] 3. Using the cDNA of wheat as a template, perform PCR amplification with the primer pair consisting of TaFIP37-7B-F (5’-TGTGGCGCAGTCTCGTGTCT-3’; as shown in SEQ ID NO.3) and TaFIP37-7B-R (5’-AACGGCAAACAGTCCCTTCAAG-3’; as shown in SEQ ID NO.4) to obtain the PCR amplification product of the full-length CDS of TaFIP37-7B. Detect the PCR product by 1.0% agarose gel electrophoresis ( Figure 1 ).

[0049] Table 1 PCR amplification system

[0050]

[0051] The PCR reaction program is: pre-denaturation at 95 °C for 30 sec; denaturation at 95 °C for 5 sec, annealing at 55 °C for 10 sec, extension at 72 °C for 1 min, with 35 cycles; extension at 72 °C for 10 min.

[0052] 4. Ligate the PCR amplification product to the cloning vector pMD18 to obtain the recombinant cloning vector pMD18-TaFIP37-7B, and transform it into Escherichia coli to pick monoclonal colonies for sequencing.

[0053] The sequencing results show that the nucleotide sequence CDS of the PCR amplification product is as shown in SEQ ID NO.2. Name the gene shown in SEQ ID NO.2 as the TaFIP37-7B gene. The TaFIP37-7B gene encodes the TaFIP37-7B protein, and the amino acid sequence of the TaFIP37-7B protein is as shown in SEQ ID NO.1.

[0054] Example 2

[0055] Overexpression of the TaFIP37-7B gene improves the drought and salt tolerance of Arabidopsis thaliana

[0056] (1) Construction of the transformation vector and Agrobacterium transformation

[0057] Using the pMD18-TaFIP37-7B recombinant vector obtained in Example 1 as a template, PCR amplification was performed using the primer pair consisting of 35s-TaFIP37-7B-F (5'-GAGAGAACACGGGGGACTCTAGAATGGCGGACCCACCTTCCCC-3'; as shown in SEQ ID NO.5) and 35s-TaFIP37-7B-R (5'-CCGCTACCACCGCTACCGAGCTCGGACTCCGCTTTTATCGGCTG-3'; as shown in SEQ ID NO.6) to obtain a PCR amplification product, and the PCR product was recovered by gel extraction. The gel-extracted product was ligated with the linearized PBI121-35S-GFP vector using In-Fusion technology to obtain the recombinant plasmid PBI121-35S-TaFIP37-7B-GFP; after verifying the correct sequence by sequencing, the plasmid of the positive bacterial solution was extracted for standby.

[0058] Table 2 PCR amplification system

[0059]

[0060] Add 1 μg of the PBI121-35S-TaFIP37-7B-GFP recombinant plasmid to 100 μl of Agrobacterium tumefaciens GV3101 competent cells, gently mix, and place on ice for 30 minutes; quickly freeze in liquid nitrogen for 10 minutes, and then heat shock in a 37°C water bath for 5 minutes; add 0.6 ml of antibiotic-free liquid LB medium, culture at 28°C with slow shaking at 180 rpm for 3 hours; then centrifuge at 4000 rpm to remove 0.6 ml of the supernatant, and suspend the remaining 100 μl of bacteria with a pipette and evenly spread them on an LB plate medium containing 50 μg / ml rifampicin and 50 μg / ml kanamycin, and culture in the dark at 28°C for 48 h; pick the transformed colonies and culture them in a liquid LB medium containing 50 μg / ml rifampicin and 50 μg / ml kanamycin and perform PCR amplification detection to obtain Agrobacterium strains containing the recombinant expression vector PBI121-35S-TaFIP37-7B-GFP;

[0061] (2) Obtaining of TaFIP37-7B transgenic Arabidopsis thaliana

[0062] Surface-sterilize the wild-type Arabidopsis thaliana seeds and sow them on MS medium for growth. After vernalization at 4°C for 2 days, place them in an incubator at a temperature of 22°C during the day / 18°C at night, with a light cycle of 16 / 8 hours and a light intensity of 7500 lux for growth. Transplant the 12-day-old seedlings into the soil for growth; when growing to the flowering stage, water the Arabidopsis thaliana plants sufficiently for use as receptor materials for transgenic transformation; the ratio of vermiculite to nutrient soil in the soil is 1:1;

[0063] Pick the Agrobacterium positive clones into LB liquid medium containing 50 μg / ml kanamycin and 50 μg / ml rifampicin, and activate them at 28°C; inoculate them into 100 ml of LB medium containing 50 μg / ml kanamycin and 50 μg / ml rifampicin at a ratio of 1:100, and culture them at 28°C with shaking at 230 rpm until the OD600 reaches 1.0 - 1.5; at room temperature, centrifuge at 4000 rpm for 10 minutes to collect the bacterial cells, discard the supernatant, and suspend the bacterial cells in the transformation buffer; after the bacterial cells are fully suspended, cut off the pods and the already opened flowers of the prepared Arabidopsis plants, immerse them in the transformation buffer for 1 minute, take them out and place them on their sides, keep them moist, and culture them in the dark for 24 hours; then straighten the plants and culture them under normal conditions until the seeds mature, and harvest the seeds for screening of the transformed homozygous lines;

[0064] (3) Screening of homozygous lines

[0065] Dry the seeds of the transformed plants harvested for one week and then sow them on the 1 / 2MS screening medium containing 40 μg / ml kanamycin (only transgenic plants can grow on the kanamycin selection medium), vernalize them at 4°C for 2 days, and place them in an incubator with a temperature of 22°C during the day / 18°C at night, a light cycle of 16 / 8 hours, and a light intensity of 7500 lux for growth; when the plants grow to 4 true leaves, transplant them into flower pots and continue to grow. When the Arabidopsis grows well, take a small amount of leaves to extract DNA and perform PCR detection according to the method in step (4) below to determine whether the Arabidopsis lines overexpressing TaFIP37-7B are successfully transformed. After maturity, harvest the T1 generation seeds by individual plants; sow the harvested T1 generation seeds by individual plants on the kanamycin selection medium as well, observe the segregation ratio of the transgenic plants, and pick the transgenic lines close to 3:1 to harvest the T2 generation seeds; sow the T2 generation seeds by individual plants on the kanamycin selection medium to obtain the T2 generation homozygous plants that no longer show kanamycin resistance segregation, and propagate and harvest the T3 generation seeds.

[0066] (4) RT-PCR detection of transgenic Arabidopsis

[0067] Take the T3 generation homozygous transgenic Arabidopsis thaliana lines (OE-1, OE-2, OE-3) of TaFIP37-7B obtained in step (3), and wild-type Arabidopsis thaliana (WT). Isolate total RNA using the Trizol method (TianGen), immediately eliminate genomic contamination using the DNAseⅠ method (Takara), and then measure the concentration using Nanodrop1000 (Thermo Scientific product, USA). Uniformly take 2 μl of the sample and run it on a 0.8% agarose gel. Take 1 μg of total RNA, use recombinant M-MLV reverse transcriptase, and use Oligo(dT) as a primer to synthesize cDNAs. Use specific primers TaFIP37-7B-F1 (5’-GACAAAGGTGGAGGAAAGTGCAC-3’; as shown in SEQ ID NO.7) and TaFIP37-7B-R1 (5’-CTGATTTAGCAGCTTCCAGTTCCAC-3’; as shown in SEQ ID NO.8) to perform PCR amplification on the cDNA of the gene TaFIP37-7B, and use the gene AtActin2 in Arabidopsis thaliana as an internal reference, with primers AtActin2-F (5’-GGTAACATTGTGCTCAGTGGTGG-3’; as shown in SEQ ID NO.9) and AtActin2-R (5’-GCATCAATTCGATCACTCAGAG-3’; as shown in SEQ ID NO.10).

[0068] Table 3 PCR identification reaction system

[0069]

[0070]

[0071] PCR reaction program: Pre-denaturation at 95℃ for 30 sec; denaturation at 95℃ for 5 sec, annealing at 57℃ for 30 sec, extension at 72℃ for 1 min, cycle 35 times; extension at 72℃ for 10 min.

[0072] Perform agarose gel electrophoresis on the PCR amplification products, and the results are as shown in Figure 2 A shown in. Further, use the TaFIP37-7B-F1 / R1 and AtActin2-F / R primer pairs to perform fluorescence quantitative analysis on the TaFIP37-7B expression level of the T3 generation transgenic Arabidopsis thaliana lines, Figure 2 The results in B show that the target gene TaFIP37-7B is not expressed in wild-type Arabidopsis thaliana plants; while the target gene TaFIP37-7B is highly expressed in the transgenic Arabidopsis thaliana lines (OE-1, OE-2, OE-3) of TaFIP37-7B.

[0073] (5)Phenotypic analysis of drought resistance of TaFIP37-7B transgenic Arabidopsis

[0074] 1) Identification of germination rate phenotype

[0075] The transgenic Arabidopsis seeds and wild-type seeds were respectively disinfected with 75% ethanol for 2 minutes in a laminar flow hood. The seeds were transferred to autoclaved filter paper with a pipette and air-dried. Then the seeds were sown on 1 / 2 MS solid medium. The solid medium without mannitol was used as the control, and the solid medium containing 200 mM mannitol was used to simulate drought stress. After vernalization for 3 days, normal cultivation was carried out (using the growth conditions in step (3)). After 7 days, the germination rates of each line were counted to identify the drought resistance of Arabidopsis lines overexpressing the gene TaFIP37-7B during the germination period.

[0076] 2) Identification of root length phenotype

[0077] The transgenic Arabidopsis seeds and wild-type seeds were respectively disinfected with 75% ethanol for 2 minutes in a laminar flow hood. The seeds were transferred to autoclaved filter paper with a pipette and air-dried. Then the seeds were sown on 1 / 2 MS solid medium. After vernalization for 3 days, normal cultivation was carried out. After 5 days, the TaFIP37-7B transgenic plants and wild-type plants on 1 / 2 MS solid medium were transplanted to 1 / 2 MS solid medium containing 200 mM mannitol to simulate drought stress for growth. When growing for 14 days, the primary root lengths of each line were measured to identify the drought resistance of each line during the early growth of seedlings.

[0078] 3) Identification of drought resistance phenotype at the seedling stage

[0079] The TaFIP37-7B transgenic plants and wild-type plants (WT) grown on MS medium were transplanted into round pots filled with a substrate of uniform compactness. After normal cultivation until the 3-week-old stage, drought treatment (i.e., stopping watering) was carried out. After 12 days, when the phenotypic differences were obvious (i.e., the wild rosette leaves were severely dried and the overexpression lines were severely wilted), rehydration was carried out. After 3 days of rehydration, the survival rates of each line were counted (plants that could resume growth and produce seeds were defined as surviving plants). The experiment was designed with 3 replicates, and the number of plants in each line was 13 for each replicate. The average value was taken for statistical analysis.

[0080] Figure 3 The results showed that the germination rate of the transgenic lines overexpressing the gene TaFIP37-7B under simulated drought stress was significantly higher than that of the wild type ( Figure 3 in A, B), the primary root length was significantly longer than that of wild-type Arabidopsis ( Figure 3 in C, D), and the survival rate after rehydration under drought stress at the seedling stage was significantly higher than that of the wild type ( Figure 3 in E, F). The above results indicated that overexpression of the gene TaFIP37-7B could improve the drought resistance of transgenic plants.

[0081] (6) Phenotypic analysis of salt tolerance of TaFIP37-7B transgenic Arabidopsis thaliana

[0082] 1) Phenotypic identification of germination rate

[0083] Sterilize transgenic Arabidopsis thaliana seeds and wild-type seeds with 75% ethanol for 2 minutes respectively in a laminar flow hood. Transfer the seeds to autoclaved filter paper with a pipette and let them air dry. Then sow the seeds on 1 / 2 MS solid medium. Use the solid medium without mannitol as the control, and the solid medium containing 150 mM sodium chloride for salt stress. After vernalization for 3 days, conduct normal cultivation (using the growth conditions in (3) of Example 2). After 7 days, count the germination rate of each line to identify the salt tolerance of Arabidopsis thaliana lines overexpressing the TaTaFIP37-7B gene during the germination period.

[0084] 2) Phenotypic identification of root length

[0085] Sterilize transgenic Arabidopsis thaliana seeds and wild-type seeds with 75% ethanol for 2 minutes respectively in a laminar flow hood. Transfer the seeds to autoclaved filter paper with a pipette and let them air dry. Then sow the seeds on 1 / 2 MS solid medium. After vernalization for 3 days, conduct normal cultivation. After 5 days, transplant the TaFIP37-7B transgenic plants and wild-type plants on 1 / 2 MS solid medium to 1 / 2 MS solid medium containing 150 mM sodium chloride for growth. Measure the primary root length of each line at 14 days of growth to identify the salt tolerance of each line during the early growth of seedlings.

[0086] Figure 4 The results show that the germination rate of transgenic lines overexpressing the TaFIP37-7B gene under salt stress is significantly higher than that of the wild type ( Figure 4 A, B in Figure 4 ), and the primary root length is significantly longer than that of wild-type Arabidopsis thaliana (

[0087] Example 4. Obtaining and phenotypic identification of wheat overexpressing the TaFIP37-7B gene

[0088] 1. Construction of TaFIP37-7B overexpression recombinant vector

[0089] Using the pMD18-TaFIP37-7B recombinant vector obtained in Example 1 as a template, PCR amplification was performed using the primer pair consisting of CUB-TaFIP37-7B-F (5’-GAAAAAGAGGGGGATTAAAGCTTATGGCGGAC CCACCTTCCCCC-3’; as shown in SEQ ID NO.11) and CUB-TaFIP37-7B-R (5’-CGCTACCACCGCTACCGAGCTCGGACTCCGCTTTTATCGGCTG-3’; as shown in SEQ IDNO.12) to obtain a PCR amplification product, and the PCR product was recovered by gel extraction. The gel-extracted product was ligated to the linearized CUB-pUbi-GFP vector using In-Fusion technology to obtain the recombinant plasmid CUB-pUbi-TaFIP37-7B-GFP( Figure 5 A); after verifying the correct sequence by sequencing, the plasmid of the positive bacterial solution was extracted for standby.

[0090] Table 4 PCR amplification system

[0091]

[0092] PCR reaction procedure: pre-denaturation at 95°C for 30 sec; denaturation at 95°C for 5 sec, annealing at 58°C for 30 sec, extension at 72°C for 1 min, 35 cycles; extension at 72°C for 10 min.

[0093] 2. Obtaining recombinant Agrobacterium tumefaciens

[0094] Add 1 μg of the CUB-pUbi-TaFIP37-7B-GFP recombinant plasmid to 100 μl of Agrobacterium tumefaciens GV3101 competent cells, gently mix, and place on ice for 30 minutes; quickly freeze in liquid nitrogen for 10 minutes, then heat shock in a 37°C water bath for 5 minutes; add 0.6 ml of antibiotic-free liquid LB medium, culture at 28°C with low-speed oscillation at 180 rpm for 3 hours; then centrifuge at 4000 rpm to remove 0.6 ml of the supernatant, suspend the remaining 100 μl of bacterial cells with a pipette and evenly spread them on an LB plate medium containing 50 μg / ml rifampicin and 50 μg / ml kanamycin, and culture in the dark at 28°C for 48 h; pick the transformed colonies and culture them in a liquid LB medium containing 50 μg / ml rifampicin and 50 μg / ml kanamycin, and perform PCR amplification detection to obtain an Agrobacterium tumefaciens strain containing the recombinant expression vector CUB-pUbi-TaFIP37-7B-GFP.

[0095] 3. Obtaining transgenic wheat

[0096] The recombinant Agrobacterium tumefaciens GV3101 / CUB-pUbi-TaFIP37-7B-GFP was transformed into the wheat variety Fielder (hereinafter also referred to as wild-type wheat) by Agrobacterium-mediated gene transformation to obtain T0 generation plants, which were planted in a 22°C greenhouse (16 h-light / 8 h-dark); specific primers TaFIP37-7B-F2 (5’-TGGGCTGTACAAACACATGGATG-3’; as shown in SEQ ID NO.13) and TaFIP37-7B-R2 (5’-GTGCAGATGAACTTCAGGGTCAG-3’; as shown in SEQ ID NO.14) were used to identify positive plants by PCR in the T0 generation plants ( Figure 5 in B), and T1 generation seeds were obtained after self-crossing; positive plants were obtained after PCR identification of the T1 generation plants, T2 generation seeds were obtained after self-crossing, positive plants were obtained after PCR identification of the T2 generation plants, and T3 generation seeds were obtained after self-crossing.

[0097] Table 5 PCR identification reaction system

[0098]

[0099] PCR reaction program: pre-denaturation at 95°C for 30 sec; denaturation at 95°C for 5 sec, renaturation at 55°C for 30 sec, extension at 72°C for 1 min, 35 cycles; extension at 72°C for 10 min.

[0100] 4. qRT-PCR detection of transgenic wheat

[0101] Take the wild-type wheat (WT) and T3 generation TaFIP37-7B transgenic wheat lines (OE-1, OE-2, OE-3) obtained in step 3, isolate total RNA by the TRIZOL method, then reverse transcribe it into cDNA, and use specific primers TaFIP37-7B-F1 (5’-GACAAAGGTGGAGGAAAGTGCAC-3’; sequence 7) and TaFIP37-7B-R1 (5’-CTGATTTAGCAGCTTCCAGTTCCAC-3’; sequence 8) to quantitatively analyze the expression level of the gene TaFIP37-7B by qRT-PCR, with the wheat TaActin1 gene as an internal reference, and the primer pair is TaActin1-F (5’-ATGTTGTTCTCAGTGGAGGTTCTAC-3’; as shown in SEQ ID NO.15) and TaActin1-R (5’-CTGTATTTCCTTTCAGGTGGTGC-3’; as shown in SEQ ID NO.16); the relative expression level is calculated using 2 -ΔΔCT Calculation. Figure 5The results of qRT-PCR showed that the expression levels of TaFIP37-7B in the T3 transgenic lines (OE-1, OE-2, OE-3) were significantly higher than those in the wild type Fielder (WT).

[0102] Table 6 Reaction system of quantitative real-time PCR

[0103]

[0104] Table 7 Reaction program of quantitative real-time PCR

[0105]

[0106] 5. Phenotypic analysis of drought resistance in transgenic wheat

[0107] The wheat seeds to be tested were the T3 seeds of OE-1, the T3 seeds of OE-2, the T3 seeds of OE-3, and the wild type Fielder seeds.

[0108] Identification of seedling survival rate:

[0109] 1. Sow 48 wheat seeds to be tested (12 seeds for each line) in a box filled with nutrient soil (48.0 cm × 15.0 cm × 12.0 cm), and cultivate them under normal conditions until the seedlings have two leaves and one heart to obtain the wheat seedlings to be tested.

[0110] 2. After completing step 1, conduct drought treatment (stop watering) for about 14 days until obvious phenotypic differences appear, that is, the leaves of the transformed receptor wheat variety Fielder are significantly withered, and the leaves of the T3 transgenic TaFIP37-7B wheat (OE-1, OE-2, and OE-3) are severely wilted.

[0111] 3. After completing step 2, re-water the plants, and observe the phenotypes and count the survival rate 3 days later (define the plants that can grow and produce seeds normally as surviving plants. The survival rate is the percentage of the number of surviving plants to the total number of plants).

[0112] Figure 6 There were no obvious differences in plant morphology among the plants in Figure A before drought treatment. Figure 6 There were significant differences in plant morphology among the plants in Figure B 3 days after re-watering after drought treatment. Compared with the wild type Fielder, the overexpression lines grew significantly better. The number of plants in the wild type Fielder that resumed growth was significantly less. The results of survival rate statistics in Figure C showed that compared with the wheat variety Fielder, the survival rates of the T3 transgenic TaFIP37-7B wheat (OE-1, OE-2, and OE-3) were significantly higher than those of the wild type wheat Fielder.

[0113] 4. Analysis of water loss rate of seedlings at the three-leaf stage

[0114] The seeds of the wild type and three overexpression lines were sown in a petri dish with double-layer filter paper. After the seeds showed white tips, they were transplanted into a seedling-growing substrate and managed with normal water and fertilizer. When they grew to the three-leaf stage, the middle section of the second leaf was cut, and the initial weight was measured. Each sample had 3 replicates. Starting in a certain order, the samples were weighed and counted using an analytical balance; the weight was measured every 30 minutes until 6 hours. The water loss at each time point was obtained by subtracting the fresh weight at each time point from the initial fresh weight, and the water loss rate at each time point was calculated by dividing the water loss at each time point by the initial fresh weight. Figure 6 The statistical analysis results of the water loss rate in D showed that, compared with the wild type, the water loss rate of the overexpression lines was significantly lower than that of the wild type.

[0115] 5. Relative water content analysis

[0116] The seeds of the wild type and three overexpression lines were sown in a petri dish with double-layer filter paper. After the seeds showed white tips, they were transplanted into a seedling-growing substrate and managed with normal water and fertilizer. When they grew to the three-leaf stage, the seedlings were divided into two groups. The control group was managed with normal water and fertilizer, and watering was stopped after thorough watering for the drought treatment. When the leaves of the wild-type plants began to wilt, the leaves were cut and placed on clean filter paper. Starting in a certain order, the fresh weight was measured using an analytical balance and recorded as m1; then the leaves were put into a 50 ml centrifuge tube, soaked in tap water for 2 hours, the water on the leaf surface was dried, and the weight was quickly measured and recorded as m2; the leaves were placed in a 60 °C drying oven for 12 hours until no water was lost. The dry weight was measured and recorded as m3.

[0117] Calculation formula: Leaf relative water content (%) = (m1 - m3) / (m2 - m3) × 100%

[0118] Figure 6 The statistical analysis results of the relative content in E showed that, compared with the wild type, there was no significant difference in the relative water content of the overexpression lines under normal conditions; after the drought treatment, the leaf relative water content of the overexpression lines was significantly higher than that of the wild type.

[0119] Based on the above results, overexpression of TaFIP37-7B can significantly improve the drought resistance of transgenic wheat.

[0120] Example 5. Phenotypic analysis of transgenic wheat salt tolerance

[0121] The wheat seeds to be tested were the T3 generation seeds of OE-1, the T3 generation seeds of OE-2, the T3 generation seeds of OE-3, and the seeds of wheat variety Fielder.

[0122] The experiment was repeated three times and the average value was taken. The steps for each repetition were as follows:

[0123] Take the wheat seeds to be tested, germinate them at room temperature for 3 days, and then transfer them to the hydroponic solution. Cultivate them at 22 °C with an alternating light and dark cycle (16 h light / 8 h dark) until the one-leaf and one-heart stage (7 days) to obtain the wheat seedlings to be tested.

[0124] The solutes and their concentrations in the hydroponic solution are 3 mM / L KH2PO4, 1 mM / L Mg SO4, 1 mM / L CaCl2·2H2O, 0.1 mM / L Fe-EDTA, 8 mM / L NH4NO3, 25 μM / L H3BO3, 2 μM / L MnSO4, 2 μM / L ZnSO4, 0.5 μM / L CuSO4, 0.5 μM / L Na2MoO4·2H2O, and the solvent is water.

[0125] 2. Transfer 10 wheat seedlings to be tested with consistent growth status to the hydroponic solution (control) or the hydroponic solution containing 200 mM NaCl, and culture them under the condition of 22 °C with alternating light and darkness (16 h light / 8 h darkness) for 14 days to observe the phenotypes. Measure and statistically analyze the plant biomass and salt tolerance coefficient (STI = measured value under salt stress treatment / measured value of the control group) of the wild-type and transgenic lines in the control group and the salt stress treatment group.

[0126] Figure 7 The results show that the dry weight and fresh weight of the transgenic lines are significantly greater than those of the wild-type under salt stress. Further calculating the salt tolerance coefficient, the salt tolerance coefficient of the transgenic lines is also significantly greater than that of the wild-type. The above results indicate that the salt tolerance of the T3 generation of TaFIP37-7B gene-transformed wheat is significantly improved.

[0127] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of the TaFIP37-7B gene or a biological material containing the TaFIP37-7B gene in any of the following: a. Used to regulate the drought resistance of plants; b. Used to regulate the salt stress tolerance of plants; c. Used for breeding drought-resistant plant varieties; d. Used for breeding salt-tolerant plant varieties; e. Used to improve the drought resistance and salt tolerance of plant germplasm resources; The nucleotide sequence of the TaFIP37-7B gene is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that: The amino acid sequence of the protein encoded by the TaFIP37-7B gene is shown in SEQ ID NO.

1.

3. The use according to claim 1 or 2, characterized in that: The biological material includes any of the following: d1, an expression cassette containing the TaFIP37-7B gene; d2, a recombinant vector containing the TaFIP37-7B gene or a recombinant vector containing the expression cassette described in d1; d3, a recombinant microorganism containing the expression cassette described in d1, or a recombinant microorganism containing the recombinant vector described in d2; d5, a transgenic plant cell line containing the expression cassette described in d1, or a transgenic plant cell line containing the recombinant vector described in d2; d6. Transgenic plant tissues and organs containing the expression cassette described in d1, or transgenic plant tissues and organs containing the recombinant vector described in d2.

4. The use according to claim 3, characterized in that: The recombinant vector is an overexpression vector.

5. The use according to claim 2, characterized in that: The application is to improve the drought resistance and salt tolerance of the recipient plant by up-regulating the expression, activity or content of the protein encoded by the TaFIP37-7B gene in the recipient plant.

6. The use according to claim 1 or 5, characterized in that: The plant is wheat or Arabidopsis thaliana.

7. A method for improving drought resistance and salt tolerance of plants, characterized in that: By up-regulating the expression, activity or content of TaFIP37-7B protein in the recipient plant, the drought resistance and salt tolerance of the recipient plant are improved; the TaFIP37-7B protein is any of the following proteins: a1), a protein encoded by the amino acids shown in SEQ ID No.1; a2) A fusion protein containing the protein described in a1).

8. The method according to claim 7, characterized in that The up-regulation of the expression, activity or content of the TaFIP37-7B protein in the recipient plant is specifically achieved by introducing the TaFIP37-7B gene into the recipient plant; the nucleotide sequence of the TaFIP37-7B gene is shown in SEQ ID NO.

2.

9. The method according to claim 7 or 8, characterized in that: The recipient plant is Arabidopsis thaliana or wheat.

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