A protein AtSRRM1L that can improve the salt tolerance of plants and its applications

Abstract

A protein AtSRRM1L that can improve the salt tolerance of plants and its application belong to the field of biotechnology. In order to improve the salt resistance of plants, the present invention provides an AtSRRM1L protein with an amino acid sequence as shown in SEQ ID NO.1. Through experiments, it is proved that the AtSRRM1L gene can respond to salt stress, and there is a physical association between AtSRRM1L and AtSnRK1. AtSnRK1 can phosphorylate AtSRRM1L, and AtSRRM1L largely depends on the kinase activity of AtSnRK1 to play its role in plant salt tolerance. This provides new clues for the new function of AtSRRM1L and its regulatory mechanism for plant salt stress tolerance.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a protein AtSRRM1L that can improve the salt tolerance of plants and its application. Background Art

[0002] Abiotic factors such as salt stress seriously affect plant growth and development, and have extremely adverse effects on agricultural production and the ecological environment. Therefore, cultivating new varieties of excellent crops that are salt-tolerant, drought-tolerant, heat-tolerant and cold-tolerant, conducting research on the stress tolerance mechanism of plants, fully exploring and improving the production potential of crops under adverse conditions, and enhancing the stress tolerance of crops are one of the major strategic issues in the development of agricultural production in China.

[0003] In order to adapt to environmental changes under abiotic stress conditions, plants have evolved a wide range of molecular mechanisms to resist the adverse effects caused by environmental changes. Serine / arginine-rich proteins in plants participate in abiotic stress responses through interactions with other splicing factors and other proteins or RNA-proteins under a complex regulatory network. The functions of many related genes with alternative splicing phenomena during plant development have been elucidated, and the role of alternative splicing in plant development and environmental responses has received increasing attention from researchers. Therefore, studying the action patterns of alternative splicing at different developmental stages and environmental conditions in plants, and clarifying its regulatory sites, will help to understand the molecular mechanism of the action of alternative splicing, and at the same time will help to more comprehensively understand the response of alternative splicing to abiotic stress and the mechanism of enhancing plant stress tolerance. Summary of the Invention

[0004] To solve the problem of how to improve the salt tolerance of plants, the present invention provides a protein AtSRRM1L that can improve the salt tolerance of plants. The protein AtSRRM1L is any one of the following proteins a) and b):

[0005] a) A protein with an amino acid sequence as shown in SEQ ID NO.1;

[0006] b) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein with an amino acid sequence as shown in SEQ ID NO.1.

[0007] In order to facilitate the purification of the protein in a), a HA tag can be connected to the amino terminus of the protein with an amino acid sequence as shown in SEQ ID NO.1.

[0008] The present invention also provides the application of the above protein AtSRRM1L in improving the salt tolerance of plants.

[0009] In one embodiment of the present invention, the application is to overexpress the protein AtSRRM1L in plants or co-express the proteins AtSRRM1L and AtSnRK1.

[0010] The present invention also provides the coding sequence of the above-mentioned protein AtSRRM1L, and the coding sequence is shown as SEQ ID NO.2.

[0011] The present invention also provides the application of the above-mentioned coding sequence in improving the salt tolerance of plants.

[0012] The present invention also provides a recombinant vector containing the above-mentioned coding sequence.

[0013] The present invention also provides a recombinant bacterium containing the above-mentioned coding sequence or the above-mentioned recombinant vector.

[0014] The present invention also provides the application of the above-mentioned recombinant vector or the above-mentioned recombinant bacterium in improving the salt tolerance of plants.

[0015] The present invention also provides a method for cultivating transgenic soybeans with salt tolerance, which is to introduce the coding gene of the protein AtSRRM1L described in claim 1 into soybeans, and the nucleotide sequence of the coding gene of the protein AtSRRM1L is shown as SEQ ID NO.2.

[0016] In one embodiment of the present invention, the transgenic soybeans are transgenic soybeans induced by Agrobacterium tumefaciens EHA105.

[0017] Advantages of the present invention:

[0018] In this invention, a serine / arginine-rich splicing factor AtSRRM1L related to plant salt stress was discovered. It is sensitive to NaCl, and qRT-PCR and GUS staining analysis showed that the AtSRRM1L gene was dominantly expressed in Arabidopsis roots, and the expression level of AtSRRM1L increased significantly after being induced by NaCl stress, and it could respond to salt stress. The AtSRRM1L protein was localized in a subnuclear structure, namely the nuclear speckle region. After being treated with NaCl, the number of nuclear speckle localizations of AtSRRM1L increased significantly. The physical association between AtSRRM1L and AtSnRK1 was confirmed by yeast two-hybrid (Y2H), bimolecular fluorescence complementation assay (BiFC), split luciferase complementation assay (SLCA), and co-immunoprecipitation (co-IP) experiments. In addition, in vitro and in vivo phosphorylation experiments determined that AtSnRK1 could phosphorylate AtSRRM1L. Salt-induced AtSnRK1-dependent phosphorylation not only promoted the localization of AtSRRM1L in nuclear speckles but also promoted the interaction with the spliceosome component AtU1-70K. Salt stress could activate AtSnRK1, and subsequent phosphorylation of AtSRRM1L by AtSnRK1 was necessary for splicing activity. Co-expression of different types of AtSnRK1 and AtSRRM1L in the 2kinm / srrm1l mutant, phenotypic analysis showed that compared with the expression of AtSnRK1(K48M) / AtSRRM1L(wt), AtSnRK1(wt) / AtSRRM1L(wt) made transgenic plants show higher salt tolerance, indicating that AtSRRM1L largely depends on the kinase activity of AtSnRK1 to perform its function. On the other hand, regardless of the kinase activity of AtSnRK1, AtSRRM1L(9A) and AtSRRM1L(9D) both had constitutive salt sensitivity and salt tolerance, indicating that AtSRRM1L has a genetic function downstream of AtSnRK1. In addition, by analyzing the phenotypes and physiological indexes of soybean plants overexpressing AtSRRM1L or AtSRRM1L(9A) under salt stress, the results showed that under salt stress, the growth of transgenic soybean plants overexpressing AtSRRM1L was better than that of AtSRRM1L(9A) transgenic soybeans. This provided new clues for the new function of AtSRRM1L and its regulatory mechanism of plant salt stress tolerance. Brief Description of the Drawings

[0019] Figure 1 It is a result diagram for qRT-PCR and GUS staining to analyze the spatio-temporal expression pattern of the AtSRRM1L gene and its response to salt stress; among them, Figure 1 A in [Figure] is a detection result diagram of the expression level of the AtSRRM1 gene in various parts of Arabidopsis plants, Figure 1Figure B shows the expression level of AtSRRM1L gene in Arabidopsis seedlings treated with 200 mM NaCl at different time periods. Figure 1 C in the figure is the expression of AtSRRM1L in transgenic Arabidopsis thaliana treated with water or 200 mM NaCl for 6 h as determined by GUS staining. SRRM1L ::GUS) expression detection results in seedlings, Figure 1 D in the figure is the quantitative analysis result of GUS activity assay;

[0020] Figure 2 Schematic diagram of the structure of AtSRRM1L protein;

[0021] Figure 3 The diagram shows the interaction between AtSnRK1 and AtSRRM1L; Figure 3 A in the figure is the result of confirming the interaction between AtSRRM1L and AtSnRK1 by yeast two-hybrid. Figure 3 Figure B is the result of BiFC confirming the interaction between AtSRRM1L and AtSnRK1. Figure 3 Figure C is the result of SLCA confirming the interaction between AtSRRM1L and AtSnRK1. Figure 3 D in the figure is the result of co-IP confirming the interaction between AtSRRM1L and AtSnRK1 in plants;

[0022] Figure 4 Figure 2 is the result of phosphorylation analysis of AtSRRM1L by AtSnRK1; Figure 4 A in the formula is Zn 2+ The results of in vitro phosphorylation analysis of AtSRRM1L by AtSnRK1 detected by Phos-tagbiotin BTL-104 antibody. Figure 4 Figure B shows the phosphorylation results of AtSRRM1L in AtSRRM1L / Col-0 and AtSRRM1L / 2kinm transgenic lines after treatment with 200 mM NaCl for 6 h;

[0023] Figure 5 This is the result of the analysis of the localization of AtSRRM1L protein in nuclear speckles;

[0024] Figure 6 The figure shows the binding results of AtSRRM1L and pre-mRNA obtained by RNA-seq analysis; Figure 6 A in the figure is the Venn diagram of AS transcripts between srrm1l mutant and wild-type plants. Figure 6 Figure B is the GO enrichment analysis result of AtSRRM1L binding to mRNA. Figure 6C in it is the verification result graph of AtSRRM1L binding to RNA in vitro RNA-EMSA experiment;

[0025] Figure 7 It is the result graph of the effect of phosphorylation of AtSnRK1 on the nuclear speckle localization of AtSRRM1L; Figure 7 A in it is the subcellular localization result graph of AtSRRM1L-GFP and AtSRRM1L(3A) / (6A) / (9A)-GFP in tobacco, Figure 7 B in it is the result graph of the effect of phosphorylation of AtSRRM1L by AtSnRK1 on subcellular localization under salt stress;

[0026] Figure 8 It is the result graph of the effect of phosphorylation of AtSRRM1L by AtSnRK1 on the alternative splicing of the precursor mRNA of the target gene NFYA10 by AtSRRM1L; Figure 8 A in it is the in vitro RNA-EMSA analysis result graph of AtSRRM1L binding to the precursor mRNA of NFYA10, Figure 8 B in it is the analysis result graph of the binding ability of SRRM1L to the precursor mRNA of NFYA10 in plant cells overexpressing GFP and SRRM1L-GFP genes under Col-0 and 2kinm backgrounds;

[0027] Figure 9 It is the result graph of the effect of phosphorylation of AtSRRM1L by AtSnRK1 on the salt tolerance of Arabidopsis under salt stress; Figure 9 A in it is the phenotype graph, root length and fresh weight analysis result graph of seedlings overexpressing GFP, SRRM1L-GFP and SRRM1L(9A)-GFP and complementing GFP, SRRM1L-GFP and SRRM1L(9A)-GFP in the srrm1l background under the wild-type plant (Col-0) background on 1 / 2MS medium with or without 200 mM NaCl, Figure 9 B in it is the phenotype, survival rate, relative chlorophyll content (RCC) result graph of plants grown by irrigating the soil with water or 250 mM NaCl solution, Figure 9 C in it is the phenotype, survival rate, relative chlorophyll content (RCC) result graph of transgenic plants co-expressing AtSnRK1, AtSRRM1L and their mutant genes in the 2kinm / srrm1l background after salt stress treatment;

[0028] Figure 10 It is the analysis result graph of soybeans heterologously expressing AtSRRM1L and AtSRRM1L(9A) genes; Figure 10 A in it is the Western blot identification result graph of overexpressing and RNAi transgenic soybeans, Figure 10Figure B shows the phenotypic comparison results of wild-type, GFP, RNAi-GmSRRM1L.1 / 2, SRRM1L(9A)-GFP, and SRRM1L-GFP transgenic soybean plants at 4 weeks old after treatment with water or 200 mM NaCl solution. Figure 10 Figure C shows the comparative analysis of malondialdehyde and chlorophyll contents in different transgenic soybean lines after salt treatment. Specific implementation manners

[0029] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained from commercial channels. For the quantitative tests in the following examples, three repeated experiments are set, and the results are averaged.

[0030] The Arabidopsis thaliana variety (Col-0 type) and Dongnong 50 (DN50) soybean seeds in the following examples can be obtained by the public from Northeast Agricultural University.

[0031] The Arabidopsis thaliana mutant seeds srrm1l-t (SALK_135314C) and nfya10 (SALK_127699C) in the following examples are T-DNA insertion mutants under the Col-0 background and are both purchased from the Arabidopsis Biological Resource Center (ABRC) ( https: / / abrc.osu.edu / ), and srrm1l-c is obtained by knocking out the AtSRRM1L gene through the CRISPR / Cas9 gene editing technology under the background of the Arabidopsis thaliana variety (Col-0 type) and can be obtained by the public from Northeast Agricultural University.

[0032] The 2kinm mutant in the following example has been disclosed in the literature "Li Q, Sun Q, Wang D, Liu YM, Zhang PM, Lu HR, Zhang Y, Zhang S, Wang AX, Ding XD, et al. (2022) Quantitative phosphoproteomics reveals the role of wild soybean GsSnRK1 as a metabolic regulator under drought and alkali stresses. J Proteomics 258:104528" and can be obtained by the public from Northeast Agricultural University.

[0033] The Arabidopsis double mutants nfya10 / srrm1l and 2kinm / srrm1l (the srrm1l mutant refers to the srrm1l-t mutant) in the following examples were prepared by genetic hybridization, and their identities were verified by genotyping. The public can obtain them from Northeast Agricultural University.

[0034] The pGADT7 and pGBKT7 vectors in the following examples were disclosed in the literature "Yu Y, Duan XB, Ding XD, Chen C, Zhu D, Yin KD, et al. (2017). A novel AP2 / ERF family transcription factor from Glycine soja, GsERF71, is a DNA binding protein that positively regulates alkaline stress tolerance in Arabidopsis. PLANT MOL BIOL 94:509-530". The public can obtain them from Northeast Agricultural University.

[0035] For the in vitro phosphorylation reaction steps in the following examples, please refer to the literature "Feng X, Feng P, Yu H, Yu X, Sun Q, Liu S, Minh TN, Chen J, Wang D, Zhang Q, et al. (2020) GsSnRK1 interplays with transcription factor GsERF7 from wild soybean to regulate soybean stress resistance. Plant Cell Environ 43:1192-1211".

[0036] The Escherichia coli competent DH5α Chemically Competent Cell, Escherichia coli prokaryotic expression competent BL21(DE3) Chemically Competent Cell, Agrobacterium tumefaciens competent GV3101 Chemically Competent Cell, EHA105 Chemically Competent Cell and Saccharomyces cerevisiae competent Y2HGold Chemically Competent Cell in the following examples were purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0037] The accession number of the AtSnRK1 gene described in the present invention is AT2G29210.

[0038] Example 1: Cloning of Arabidopsis Splicing Factor AtSRRM1L Gene Rich in Serine / Arginine and Analysis of Its Expression Pattern

[0039] I. Treatment of Plant Materials

[0040] 1. Treatment of Arabidopsis Seeds

[0041] Arabidopsis seeds were surface-sterilized with 1% NaClO and then sown on 1 / 2MS medium (1 / 2 Murashige-Skoog salts, 2% sucrose, 0.8% agar, pH 5.7). After vernalization at 4°C in the dark for 3 days, the seeds were placed in a light incubator with a photoperiod of 16 h for vertical growth. Pot-grown seedlings were cultivated by sowing Arabidopsis seeds in pots containing vermiculite and nutrient soil at a ratio of 3:1. For the germination test, vernalized seeds were sown on 1 / 2MS medium containing 0 mM or 200 mM NaCl, and the seed germination rate was counted for 5 days. Each genotype was subjected to three biological replicates. For salt stress treatment, seedlings that had been normally cultivated in pots for 25 days were irrigated with Hoagland's nutrient solution containing 250 mM NaCl for 10 days, and the culture solution was replaced regularly during this period. Before the stress treatment of the 2kinm mutant and the double mutant 2kinm / srrmll, the mutant plants that had grown for 20 days were sprayed with 10 μM β-estradiol to reduce the expression of the SnRK1.2 gene in the plants.

[0042] 2. Treatment of Soybean Seeds

[0043] Select healthy and mature soybean seeds (DN50), and use the chlorine disinfection method [100 mL of sodium hypochlorite (8% effective concentration) plus 5 mL of concentrated hydrochloric acid] for continuous sterilization for 16 - 20 h. This experimental process is carried out in a fume hood. After sterilization, the seeds are blown in a laminar flow hood for 24 h to disperse the residual chlorine. The culture dish containing the sterilized soybeans is sealed and stored at 4°C for later use.

[0044] II. RNA Extraction

[0045] Arabidopsis seedlings that had grown in soil for 25 days were transferred to Hoagland's nutrient solution for 1 day and then transferred to Hoagland's culture solution containing 200 mM NaCl. Samples were taken after treatment for 0 h, 3 h, 6 h, 12 h, and 24 h respectively. Samples were also taken from the roots, stems, leaves, and flowers of Arabidopsis plants that had grown for 40 days. The total RNA was extracted according to the instructions of the PlantRNAKit (OMEGA) kit.

[0046] III. Obtaining cDNA

[0047] Using the total RNA obtained in Step 2 above as a template, cDNA was synthesized according to a reverse transcription kit (TOYOBO).

[0048] IV. PCR Amplification

[0049] Using the cDNA obtained in Step 3 as a template, PCR amplification was performed using the primers AtSRRM1L-Clone-F / R (SEQ ID NO.3 and SEQ ID NO.4) and the PrimeSTAR Max DNA Polymerase (TaKaRa) kit to obtain a PCR product. The PCR product was electrophoretically detected using 1% agarose gel. The electrophoresis result showed that the band size was about 2.6 kb. The PCR product was recovered using an agarose gel recovery kit (TRANSGEN BIOTECH), and the purified recovered product was sequenced. The sequencing result showed that the amplified product obtained by PCR amplification was 2637 bp in size, and its nucleotide sequence was as shown in SEQ ID NO.2, which was named the AtSRRM1L gene. The amino acid sequence of the protein encoded by the AtSRRM1L gene was as shown in SEQ ID NO.1.

[0050] V. Real-time Fluorescent Quantitative PCR Analysis of the Expression Pattern of AtSRRM1L

[0051] qRT-PCR amplification was performed on the cDNA of different tissues of Arabidopsis thaliana plants grown for 40 d and the cDNA of Arabidopsis thaliana seedlings grown for 25 d treated with 2 m M NaCl for 0 h, 3 h, 6 h, 12 h, and 24 h using the primers AtSRRM1L-qPCR-F / R (SEQ ID NO.5 and SEQ ID NO.6). According to the qRT-PCR results, the AtSRRM1L gene was expressed in all organs of Arabidopsis thaliana plants, and the expression level was relatively high in roots (see A in Figure 1 ). Wild-type Arabidopsis thaliana seedlings were treated with 200 mM NaCl, and after a certain period of time, materials were collected to extract total RNA. The results showed that the transcriptional level of AtSRRM1L was upregulated by salt induction (see B in Figure 1 ).

[0052] VI. GUS Activity Staining and GUS Content Determination to Analyze the Expression Pattern of AtSRRM1L under NaCl Stress

[0053] Take Arabidopsis thaliana grown on 1 / 2 MS medium for 5 days old Pro SRRM1L::After treating GUS transgenic seedlings with 200 mM NaCl for 6 h, GUS staining was performed. The Arabidopsis seedlings of the control and those treated with NaCl were respectively placed in GUS staining solution (100 mM Na3PO4, pH 7.0, 10 mM EDTA, 2 mM K3Fe(CN)6, 2 mM K4Fe(CN)6, 0.1% Triton X-100 and 1 mM X-Gluc), incubated overnight at 37 °C, and then the seedlings were decolorized with 70% ethanol. After removing the excess staining solution, the seedlings were transferred to ddH2O, observed and photographed. Reagents required for quantitative detection of GUS activity in plants: (1) 0.1 M phosphate buffer (pH 7.0); (2) 10% SDS solution; (3) 0.5 M EDTA (pH 8.0); (4) GUS enzyme extraction solution; (5) MUG substrate; (6) Stop Buffer (0.2 M Na2CO3); (7) Coomassie Brilliant Blue G250 solution; (8) 1 mg / mL BSA. Total protein was extracted using GUS extraction solution, and the Bradford method was used to determine the total protein concentration. Take 100 μL of the total protein extraction solution, add it to 400 μL of GUS extraction buffer at 37 °C, then add 500 μL of MUG substrate, and place it in an incubator at 37 °C. Every 15 min, take 200 μL of the mixed reactant and add it to 800 μL of reaction termination solution, and sample 5 times in total, and store it in the dark at room temperature. Use a fluorescence spectrophotometer to measure the fluorescence intensity values at each time point with an excitation wavelength of 365 nm and an emission wavelength of 455 nm. Make a curve of the fluorescence intensity value against the reaction time, calculate the change value of the fluorescence intensity per unit time, and calculate the GUS activity (nmol 4-MU / min mg protein). The results show that Pro SRRM1L ::After salt stress treatment of GUS transgenic Arabidopsis seedlings, the GUS activity of the plants was significantly up-regulated. These results indicate that the AtSRRM1L gene responds to salt stress. (See Figure 1 C in Figure 1 and D in

[0054] ). The amino acid sequence of the AtSRRM1L protein was analyzed using the SMART online software. The results showed that the AtSRRM1L protein contains a conserved PWI domain, belonging to the PWI superfamily, and a serine / arginine-rich region (RS region) (see Figure 2 ).

[0055] Example 2: Interaction between AtSnRK1 and AtSRRM1L

[0056] I. Yeast two-hybrid verification of the interaction between AtSnRK1 and AtSRRM1L

[0057] (1) Construction of pGBKT7-AtSnRK1 and pGADT7-AtSRRM1L expression vectors

[0058] 1. Obtaining the AtSnRK1 gene

[0059] Using Arabidopsis thaliana total cDNA as a template, PCR amplification was performed with the primers pGBKT7-AtSnRK1-SmaI SalIF / R (SEQ ID NO.7 and SEQ ID NO.8) to obtain a PCR amplification product, which is the AtSnRK1 gene with SmaI and SalI restriction enzyme sites and homologous arms to the vector part.

[0060] 2. Construction of the recombinant vector pGBKT7-AtSnRK1

[0061] The pGBKT7 vector was double digested with the restriction enzymes SmaI and SalI, and the AtSnRK1 gene obtained in step 1 was ligated to the double-digested pGBKT7 vector to obtain the pGBKT7-AtSnRK1 recombinant vector, and the pGBKT7-AtSnRK1 recombinant vector was sequenced and verified.

[0062] The sequencing results showed that the pGBKT7-AtSnRK1 recombinant vector is a vector obtained by replacing the DNA fragment between the SmaI and SalI restriction enzyme sites of the pGBKT7 vector with the AtSnRK1 gene, and keeping the other sequences of the pGBKT7 vector unchanged. The pGBKT7-AtSnRK1 recombinant vector expresses the AtSnRK1 protein.

[0063] 3. Construction of the recombinant vector pGADT7-AtSRRM1L

[0064] Using Arabidopsis thaliana total cDNA as a template, PCR amplification was performed with the primers pGADT7-AtSRRM1L-SmaIF / R (SEQ ID NO.9 and SEQ ID NO.10) to obtain a PCR amplification product, which is the AtSRRM1L gene with SmaI restriction enzyme site and homologous arms to the vector part. The pGADT7 vector was digested with the restriction enzyme SmaI, and after the digestion product was recovered and purified by gel electrophoresis, it was ligated to the above PCR product to obtain the pGADT7-AtSRRM1L recombinant vector, and the pGADT7-AtSRRM1L recombinant vector was sequenced and verified.

[0065] Sequencing results showed that the recombinant vector pGADT7-AtSRRM1L was obtained by inserting the SmaI restriction site of the pGADT7 vector into the AtSRRM1L gene through homologous recombination, while keeping other sequences of the pGADT7 vector unchanged. The recombinant vector pGADT7-AtSRRM1L expresses the AtSRRM1L protein.

[0066] (II) Transformation of yeast Y2HGold

[0067] According to the Y2HGold transformation instructions, the recombinant plasmids pGBKT7-AtSnRK1 and pGADT7-AtSRRM1L were co-transformed into the Y2HGold strain and spread on SD / -Trp-Leu medium, and grown in an incubator at 30 °C for 48 - 96 h. The cells were separately inoculated on solid media of SD / -Trp-Leu and SD / -Trp-Leu-His (containing 20 mM 3-AT) to analyze the interaction between AtSnRK1 and AtSRRM1L. The combinations of pGBKT7-Empty / pGADT7-AtSRRM1L and pGBKT7-AtSnRK1 / pGADT7-Empty were used as blank controls, the combination of pGBKT7-Empty / pGADT7-Empty was used as a negative control, and the combination of pGBKT7-GsSnRK1α / pGADT7-GsSnRK1β was used as a positive control.

[0068] The results were as Figure 3 shown in A below. Yeast strains containing each combination could grow on SD / -Trp-Leu medium, indicating successful co-transformation of the plasmids. However, the results of yeast growth on solid medium of SD / -Trp-Leu-His (containing 20 mM 3-AT) showed that only the yeast strain co-transformed with pGBKT7-AtSnRK1 and pGADT7-AtSRRM1L plasmids could grow, while the blank control and negative control groups could not grow, which indicated that there was an interaction between AtSnRK1 and AtSRRM1L proteins.

[0069] II. Transient transformation of tobacco to verify the interaction and localization of AtSnRK1 and AtSRRM1L

[0070] (I) Construction of pSPYNE-AtSnRK1 and pSPYCE-AtSRRM1L expression vectors

[0071] 1. Construction of the recombinant vector pSPYNE-AtSnRK1

[0072] Using the recombinant plasmid pGBKT7-AtSnRK1 as a template, PCR amplification was performed with the primers pSPYNE-AtSnRK1-KpnIF / R (SEQ ID NO.11 and SEQ ID NO.12) to obtain a PCR amplification product, which is the AtSnRK1 gene with a KpnI restriction site and a homologous arm to the vector part. The pSPYNE vector was digested with the restriction enzyme KpnI, and after the digestion product was recovered and purified by gel extraction, it was ligated with the above PCR product to obtain the recombinant vector pSPYNE-AtSnRK1, and the pSPYNE-AtSnRK1 recombinant vector was verified by sequencing.

[0073] The sequencing results showed that the recombinant vector pSPYNE-AtSnRK1 was obtained by inserting the KpnI restriction site of the pSPYNE vector into the AtSnRK1 gene through homologous recombination while keeping other sequences of the pSPYNE vector unchanged. The recombinant vector pSPYNE-AtSnRK1 expresses the AtSnRK1 protein.

[0074] 2. Construction of the recombinant vector pSPYCE-AtSRRM1L

[0075] Using the recombinant plasmid pGADT7-AtSRRM1L as a template, PCR amplification was performed with the primers pSPYCE-AtSRRM1L-KpnIF / R (SEQ ID NO.13 and SEQ ID NO.14) to obtain a PCR amplification product, which is the AtSRRM1L gene with a KpnI restriction site and a homologous arm to the vector part. The pSPYCE vector was digested with the restriction enzyme KpnI, and after the digestion product was recovered and purified by gel extraction, it was ligated with the above PCR product to obtain the recombinant vector pSPYCE-AtSRRM1L, and the pSPYCE-AtSRRM1L recombinant vector was verified by sequencing.

[0076] The sequencing results showed that the recombinant vector pSPYCE-AtSRRM1L was obtained by inserting the KpnI restriction site of the pSPYCE vector into the AtSRRM1L gene through homologous recombination while keeping other sequences of the pSPYCE vector unchanged. The recombinant vector pSPYCE-AtSRRM1L expresses the AtSRRM1L protein.

[0077] (2) Construction of the expression vectors pCAMBIA1300-cLUC-AtSnRK1 and pCAMBIA1300-nLUC-AtSRRM1L

[0078] The construction of the expression vectors pCAMBIA1300-cLUC-AtSnRK1 and pCAMBIA1300-nLUC-AtSRRM1L was carried out according to the method in (1) above.

[0079] (III) Transient transformation of tobacco

[0080] The bimolecular fluorescence complementation (BiFC) assay was used to detect the interaction between AtSnRK1 and AtSRRM1L. After the recombinant plasmids pSPYNE-AtSnRK1 and pSPYCE-AtSRRM1L were separately transformed into Agrobacterium tumefaciens GV3101, they were transiently co-expressed in tobacco leaves. After the tobacco plants were cultured for 2 days, the fluorescence signals of the interacting proteins were detected using a confocal laser microscope. The results are as shown in Figure 3 panel B. When the AtSnRK1-nYFP and AtSRRM1L-cYFP fusion proteins were transiently co-expressed in tobacco leaf cells, strong YFP fluorescence signals were observed in the nucleus, while co-expression with the empty vector did not show any recognizable YFP fluorescence signals, indicating that the interaction between AtSnRK1 and AtSRRM1L mainly occurs in the nucleus.

[0081] For the luciferase complementation assay (LCA), after the recombinant plasmids pCAMBIA1300-cLUC-AtSnRK1 and pCAMBIA1300-nLUC-AtSRRM1L were separately transformed into Agrobacterium tumefaciens GV3101, they were transiently co-expressed in tobacco leaves. The GsSnRK1α-cLUC / GsSnRK1β-nLUC combination was used as a positive control. After culturing for 3 days, the tobacco leaves were sprayed with a luciferin solution (1 mM luciferin, 0.01% Triton X-100), and the luciferase activity was observed using a chemiluminescent imaging system after a 20-min dark treatment. The results are as shown in Figure 3 panel C. When AtSnRK1-cLUC and AtSRRM1L-nLUC were co-infiltrated into tobacco leaves, strong luciferase activity was observed, indicating that the AtSRRM1L protein interacts with the AtSnRK1 protein.

[0082] (IV) Extraction of tobacco proteins and Western blot detection

[0083] Total proteins were extracted after the relevant plasmids were transiently expressed in tobacco leaves, and the co-immunoprecipitation (co-IP) technique was used to analyze their interaction. HA-AtSRRM1L was immunoprecipitated (i.e., IP) from all lysates using anti-HA, and the Myc-AtSnRK1 protein was detected by Western blot using an anti-Myc antibody; and Myc-AtSnRK1 was immunoprecipitated from all lysates using anti-Myc, and the HA-AtSRRM1L protein was detected by Western blot using an anti-HA antibody. The results are as shown in Figure 3As shown in D in [reference], the results indicate that there is an interaction between the AtSRRM1L protein and the AtSnRK1 protein, and they can form a protein complex.

[0084] III. Phosphorylation analysis of AtSRRM1L by AtSnRK1

[0085] (I) Construction of protein expression vectors

[0086] 1. Construction of recombinant vectors pET28a-AtSnRK1(T175E) and pET28a-AtSnRK1(K48M)

[0087] 1) Obtaining the AtSnRK1(T175E) gene

[0088] We replaced the base ACA encoding the 175th amino acid in the AtSnRK1 gene sequence with GAA, causing the 175th amino acid of the AtSnRK1 protein to mutate from threonine (T) to glutamate (E). We re-synthesized the mutated AtSnRK1 gene and named it AtSnRK1(T175E). The AtSnRK1(T175E) protein encoded by the AtSnRK1(T175E) gene has the function of phosphorylation.

[0089] Using the AtSnRK1(T175E) gene as a template, PCR amplification was performed with the primers pET28a-Myc-AtSnRK1(T175E)-SalIF / R (SEQ ID NO.15 and SEQ ID NO.16) to obtain the PCR amplification product, which is the AtSnRK1(T175E) gene with SalI restriction enzyme sites and homologous arms with the vector part.

[0090] 2) Obtaining the AtSnRK1(K48M) gene

[0091] [[ID=2)4]]We replaced the base AAG encoding the 48th amino acid in the AtSnRK1 gene sequence with ATG, causing the 48th amino acid of the AtSnRK1 protein to mutate from lysine (K) to methionine (M). We re-synthesized the mutated AtSnRK1 gene and named it AtSnRK1(K48M). The AtSnRK1(K48M) protein encoded by the AtSnRK1(K48M) gene does not have the function of phosphorylation.

[0092] Using the AtSnRK1(K48M) gene as a template, PCR amplification was carried out with the primers pET28a-Myc-AtSnRK1(K48M)-SalIF / R (SEQ ID NO.15 and SEQ ID NO.16), and the PCR amplification product was obtained, namely the AtSnRK1(K48M) gene with SalI restriction enzyme sites and homologous arms with the vector part.

[0093] 3) Construction of recombinant vectors pET28a-AtSnRK1(T175E) and pET28a-AtSnRK1(K48M)

[0094] The pET28a vector was digested with the restriction enzyme SalI. After the digestion products were recovered and purified by gel, they were respectively ligated with the above PCR products to obtain the recombinant vectors pET28a-AtSnRK1(T175E) and pET28a-AtSnRK1(K48M). The recombinant vectors pET28a-AtSnRK1(T175E) and pET28a-AtSnRK1(K48M) were respectively sequenced for verification.

[0095] The sequencing results showed that: the recombinant vector pET28a-AtSnRK1(T175E) was obtained by replacing the DNA fragment between the SalI restriction enzyme sites of the pET28a vector with the AtSnRK1(T175E) gene and keeping the other sequences of the pET28a vector unchanged. The recombinant vector pET28a-AtSnRK1(T175E) expressed the AtSnRK1(T175E) protein. The recombinant vector pET28a-AtSnRK1(K48M) was obtained by replacing the DNA fragment between the SalI restriction enzyme sites of the pET28a vector with the AtSnRK1(K48M) gene and keeping the other sequences of the pET28a vector unchanged. The recombinant vector pET28a-AtSnRK1(K48M) expressed the AtSnRK1(K48M) protein.

[0096] 2. Construction of recombinant vector pET28a-AtSRRM1L

[0097] 1) Obtaining of the AtSRRM1L gene

[0098] Using the pGADT7-AtSRRM1L recombinant plasmid as a template, PCR amplification was carried out with the primers pET28a-HA-AtSRRM1L-SalIF / R (SEQ ID NO.17 and SEQ ID NO.18), and the PCR amplification product was obtained, namely the AtSRRM1L gene with Sal I restriction enzyme sites and homologous arms with the vector part.

[0099] 2) Construction of the recombinant vector pET28a-AtSRRM1L

[0100] The pET28a vector was digested with the restriction endonuclease SalI. After the digestion product was recovered and purified by gel extraction, it was ligated with the above PCR product to obtain the recombinant vector pET28a-AtSRRM1L. The recombinant vector pET28a-AtSRRM1L was verified by sequencing.

[0101] The sequencing results showed that: the recombinant vector pET28a-AtSRRM1L was a vector obtained by replacing the DNA fragment between the SalI cleavage sites of the pET28a vector with the AtSRRM1L gene, while keeping other sequences of the pET28a vector unchanged. The recombinant vector pET28a-AtSRRM1L expressed the AtSRRM1L protein.

[0102] 3. Construction of the recombinant vector pET28a-AtSRRM1L(9A)

[0103] 1) Obtaining the AtSRRM1L(9A) gene

[0104] According to the results of phosphorylation mass spectrometry analysis, we obtained that there were nine phosphorylation sites on the AtSRRM1L protein that could be recognized by AtSnRK1, and their positions were Ser27, Thr41, Thr168, Ser285, Ser287, Ser391, Ser393, Ser41, and Ser558. Therefore, we replaced all the bases encoding these 9 amino acids in the AtSRRM1L gene sequence with GCT, so that the amino acids at 9 positions of the AtSRRM1L protein were all mutated to alanine (A). We re-synthesized the mutated AtSRRM1L gene and named it AtSRRM1L(9A). The AtSRRM1L(9A) protein encoded by the AtSRRM1L(9A) gene did not have the ability to be phosphorylated by the AtSnRK1 protein.

[0105] Using the AtSRRM1L(9A) gene as a template, PCR amplification was carried out with the primers pET28a-HA-AtSRRM1L(9A)-SalIF / R (SEQ ID NO.17 and SEQ ID NO.18) to obtain the PCR amplification product, that is, the AtSRRM1L(9A) gene with SalI cleavage sites and homologous arms to the vector part.

[0106] 2) Construction of the recombinant vector pET28a-AtSRRM1L(9A)

[0107] The pET28a vector was digested with the restriction endonuclease SalI. After the digestion products were purified by gel extraction, they were ligated with the above PCR products to obtain the recombinant vector pET28a-AtSRRM1L(9A), and the pET28a-AtSRRM1L(9A) recombinant vector was verified by sequencing.

[0108] The sequencing results showed that the pET28a-AtSRRM1L(9A) recombinant vector was a vector obtained by replacing the DNA fragment between the SalI digestion sites of the pET28a vector with the AtSRRM1L(9A) gene, while keeping other sequences of the pET28a vector unchanged. The pET28a-AtSRRM1L(9A) recombinant vector expressed the AtSRRM1L(9A) protein.

[0109] 4. Construction of recombinant vectors pET28a-AtSRRM1L(S27), pET28a-AtSRRM1L(T41), pET28a-AtSRRM1L(T168), pET28a-AtSRRM1L(S285), pET28a-AtSRRM1L(S287), pET28a-AtSRRM1L(S391), pET28a-AtSRRM1L(S393), pET28a-AtSRRM1L(S411) and pET28a-AtSRRM1L(S558)

[0110] The acquisition of the AtSRRM1L(S27), AtSRRM1L(T41), AtSRRM1L(T168), AtSRRM1L(S285), AtSRRM1L(S287), AtSRRM1L(S391), AtSRRM1L(S393), AtSRRM1L(S411) and AtSRRM1L(S558) genes and the construction of the recombinant vectors pET28a-AtSRRM1L(S27), pET28a-AtSRRM1L(T41), pET28a-AtSRRM1L(T168), pET28a-AtSRRM1L(S285), pET28a-AtSRRM1L(S287), pET28a-AtSRRM1L(S391), pET28a-AtSRRM1L(S393), pET28a-AtSRRM1L(S411) and pET28a-AtSRRM1L(S558) were carried out according to the method in step 3.

[0111] (II) Expression and purification of proteins

[0112] The protein expression vectors obtained in (i) were separately transformed into competent Escherichia coli BL21(DE3) to obtain BL21(DE3) Escherichia coli containing the above-mentioned protein expression vectors, and protein expression was induced. The expressed proteins were separately purified. The purification of AtSnRK1(T175E) and AtSnRK1(K48M) proteins was carried out using the Myc fusion protein purification kit provided by Shanghai Guyan Industrial Co., Ltd. The specific steps are detailed in the kit instructions; the purification of AtSRRM1L, AtSRRM1L(9A), AtSRRM1L(S27), AtSRRM1L(T41), AtSRRM1L(T168), AtSRRM1L(S285), AtSRRM1L(S287), AtSRRM1L(S391), AtSRRM1L(S393), AtSRRM1L(S411) and AtSRRM1L(S558) proteins was carried out using the Kangwei Century His-Tagged Protein Purification Kit, and the specific steps are detailed in the kit instructions.

[0113] (iii) Use Zn 2+ Phos-tag Biotin BTL-104 antibody kit to detect the in vitro phosphorylation of AtSRRM1L by AtSnRK1

[0114] Use Zn 2+ Phos-tag Biotin BTL-104 antibody was used to detect the phosphorylation levels of AtSRRM1L by AtSnRK1(T175E) and AtSnRK1(K49M); the phosphorylation levels of AtSRRM1L(9A), AtSRRM1L(S27), AtSRRM1L(T41), AtSRRM1L(T168), AtSRRM1L(S285), AtSRRM1L(S287), AtSRRM1L(S391), AtSRRM1L(S393), AtSRRM1L(S411) and AtSRRM1L(S558) by AtSnRK1(T175E). The specific operation steps are detailed in the Zn 2+ Phos-tag Biotin BTL-104 antibody kit instructions. The results are as Figure 4As shown in A in [reference], AtSnRK1 (T175E) phosphorylates AtSRRM1L, while AtSnRK1 (K49M) does not phosphorylate AtSRRM1L. AtSnRK1 (T175E) does not phosphorylate AtSRRM1L(9A), AtSRRM1L(S27), AtSRRM1L(T41), AtSRRM1L(T168), AtSRRM1L(S285), AtSRRM1L(S287), AtSRRM1L(S391), AtSRRM1L(S393), AtSRRM1L(S411), and AtSRRM1L(S558). It is demonstrated that the AtSnRK1 protein has a phosphorylation effect on the AtSRRM1L protein, and the 9 phosphorylation sites on the AtSRRM1L protein are the phosphorylation sites recognized by AtSnRK1.

[0115] (IV) Detection of the phosphorylation of AtSRRM1L by AtSnRK1 using Western blot

[0116] The AtSRRM1 gene was overexpressed in the wild-type (Col-0) or 2kinm background respectively. After treating the overexpressing plants HA-AtSRRM1L / Col-0 and HA-SRRM1L / 2kinm with NaCl, the phosphorylation level of AtSRRM1L in vivo was detected.

[0117] The results are as Figure 4 shown in B in [reference]. The HA-SRRM1L protein was immunoprecipitated from HA-AtSRRM1L / Col-0 and HA-SRRM1L / 2kinm plants using the HA antibody. It was detected by the Phos-tag antibody that AtSRRM1L can be phosphorylated by AtSnRK1 in vivo, and salt stress can enhance the phosphorylation level of AtSRRM1L. This indicates that the phosphorylation of AtSRRM1L by AtSnRK1 responds to salt stress.

[0118] Example 3: Subcellular localization analysis of the AtSRRM1L protein

[0119] 1. Construction of the pBWA(V)BS-3HA-AtSRRM1L-GFP expression vector

[0120] 1) Obtaining the AtSRRM1L gene

[0121] Using the above pGADT7-AtSRRM1L plasmid as a template, PCR amplification was performed with the primer pair pBWA(V)BS-3HA-SRRM1L-GFP-SmaIF / R (SEQ ID NO.19 and SEQ ID NO.20) and the PrimeSTAR Max DNA Polymerase kit to obtain a PCR amplification product, namely the AtSRRM1L gene with SmaI restriction sites and homologous arms to the vector part.

[0122] 2) Construction of the recombinant vector pBWA(V)BS-3HA-AtSRRM1L-GFP

[0123] After single digestion of the pBWA(V)BS-3HA-GFP vector with the restriction enzyme SmaI, AtSRRM1L was ligated to the digested and purified vector using homologous recombinase, and the other sequences of the pBWA(V)BS-3HA-GFP vector were kept unchanged to obtain the pBWA(V)BS-3HA-AtSRRM1L-GFP recombinant vector, and the pBWA(V)BS-3HA-AtSRRM1L-GFP recombinant vector was verified by sequencing.

[0124] The sequencing results showed that the pBWA(V)BS-3HA-AtSRRM1L-GFP recombinant vector was obtained by inserting the AtSRRM1L gene after single digestion of the pBWA(V)BS-3HA-GFP vector with SmaI and keeping the other sequences of the pBWA(V)BS-3HA-GFP vector unchanged.

[0125] 2. Construction of the pBWA(V)BS-3HA-RS SRRM1L -GFP expression vector

[0126] 1) Obtaining of the RS SRRM1L gene

[0127] Using the above pGADT7-AtSRRM1L plasmid as a template, PCR amplification was performed with the primer pair pBWA(V)BS-3HA-RS SRRM1L -GFP-SmaIF / R (SEQ ID NO.21 and SEQ ID NO.22) and the PrimeSTAR Max DNA Polymerase kit to obtain a PCR amplification product, namely the RS SRRM1L gene with SmaI restriction sites and homologous arms to the vector part.

[0128] 2) Construction of the recombinant vector pBWA(V)BS-3HA-RS SRRM1L -GFP

[0129] After single digestion of the pBWA(V)BS-3HA-GFP vector with the restriction endonuclease SmaI, the RS was then ligated to the digested and purified vector using homologous recombinase, and the other sequences of the pBWA(V)BS-3HA-GFP vector were kept unchanged to obtain the vector pBWA(V)BS-3HA-RS SRRM1L -GFP recombinant vector. The pBWA(V)BS-3HA-RS SRRM1L -GFP recombinant vector was verified by sequencing. SRRM1L The sequencing results showed that the pBWA(V)BS-3HA-RS

[0130] -GFP recombinant vector was obtained by single digestion of the pBWA(V)BS-3HA-GFP vector with SmaI and insertion of the RS SRRM1L gene, while keeping the other sequences of the pBWA(V)BS-3HA-GFP vector unchanged. SRRM1L

[0131] 3. Construction of the pBWA(V)BS-3HA-PWI SRRM1L -GFP expression vector

[0132] 1) Obtaining of the PWI SRRM1L gene

[0133] Using the above pGADT7-AtSRRM1L plasmid as a template, PCR amplification was performed with the primer pair pBWA(V)BS-3HA-PWI SRRM1L -GFP-SmaIF / R (SEQ ID NO.19 and SEQ ID NO.23) and the PrimeSTAR Max DNA Polymerase kit to obtain the PCR amplification product, that is, the PWI SRRM1L gene with SmaI restriction sites and partial homologous arms to the vector.

[0134] 2) Construction of the recombinant vector pBWA(V)BS-3HA-PWI SRRM1L -GFP

[0135] After single digestion of the pBWA(V)BS-3HA-GFP vector with the restriction endonuclease SmaI, the PWI SRRM1L was then ligated to the digested and purified vector using homologous recombinase, and the other sequences of the pBWA(V)BS-3HA-GFP vector were kept unchanged to obtain the vector pBWA(V)BS-3HA-PWI SRRM1L -GFP recombinant vector. The pBWA(V)BS-3HA-PWI SRRM1L -GFP recombinant vector was verified by sequencing.

[0136] The sequencing results showed that pBWA(V)BS-3HA-PWI SRRM1L -GFP recombinant vector was obtained by inserting the PWI gene into the pBWA(V)BS-3HA-GFP vector after single digestion with SmaI, while keeping other sequences of the pBWA(V)BS-3HA-GFP vector unchanged. SRRM1L

[0137] 4. Transient transformation of tobacco

[0138] For subcellular localization analysis, the plasmids pBWA(V)BS-3HA-GFP and recombinant plasmids pBWA(V)BS-3HA-AtSRRM1L-GFP, pBWA(V)BS-3HA-RS SRRM1L -GFP and pBWA(V)BS-3HA-PWI SRRM1L -GFP were separately transformed into Agrobacterium tumefaciens GV3101 and transiently expressed in tobacco leaves. After the tobacco was cultured for 2 days, the fluorescence signals of the interacting proteins were detected using a confocal laser microscope. The results are as Figure 5 shown. The full-length AtSRRM1-GFP was localized in the nucleus in a typical speckled pattern. PWI-GFP was present in both the cytoplasm and the nucleus, indicating that there was no any subcellular targeting signal. The RS-GFP localization signal was only present in the nucleus. Additionally, RS-GFP was localized in nuclear speckles, suggesting that both the speckle targeting and speckle retention signals were located in the RS region. These results indicate that the RS region not only contains an independent functional nuclear localization signal, but also the speckle localization signal is located in the RS region, which is necessary and sufficient for speckle localization.

[0139] Example 4: AtSRRM1L regulates alternative splicing of stress-related genes

[0140] To clarify the role of AtSRRM1L in the salt stress response of Arabidopsis thaliana, transcriptome sequencing was performed on wild-type Arabidopsis thaliana and srrm1l mutant seedlings grown for 25 days to detect whether the mutation of SRRM1L would lead to alternative splicing of stress-related genes in plants. The RNA-seq results are as Figure 6 shown in A of it. There were a total of 22,477 transcripts with alternative splicing in the srrm1l mutant, while there were 21,915 alternative spliceosomes in the wild type, and a total of 23,342 transcripts generated by AS in both the srrm1l mutant and the WT. GO enrichment analysis was performed on the genes with alternative splicing dependent on SRRM1L. The results are as Figure 6 shown in B of it. These genes with alternative splicing were enriched in response to various stresses, which means that the genes with alternative splicing events may be related to the salt stress response regulated by SRRM1L.

[0141] RNA-seq analysis showed that intron retention occurred in a total of 156 genes in the srrm1l mutant, and the effect of SRRM1L on intron retention (IR) raised the question of whether SRRM1L is an RNA-binding protein. To identify a putative SRRM1L-binding motif, MEME searches were performed using introns (the entire intron sequence plus 50 bases of the 5' and 3' flanking exon sequences of the intron sequence). The most significantly enriched motif found by the search was a CU-rich 21-bp sequence that occurred in the exon sequences flanking the 5' or 3' ends of the intron sequence and rarely within the intron, and this motif was predicted to be a potential binding site for SRRM1L. Subsequently, electrophoretic mobility shift assays (REMSA) were performed using biotinylated RNA probes synthesized based on a 21-nt sequence (5'-CUCACCUGGUAAACUCUCUCA-3') from the 5′ flanking region of intron I of AT5G06510, and the sequence of this probe was as shown in SEQ ID NO.46. The REMSA results were as Figure 6 shown in C of , SRRM1L impeded the migration of the probe, and as the protein concentration increased, the hindrance of probe migration increased. Additionally, with the addition of unlabeled probes, this migration was weakened or even abolished. These observations strongly suggest the binding of SRRM1L to the target mRNA.

[0142] Example 5: Effect of phosphorylation of AtSnRK1 on the nuclear speckle localization of AtSRRM1L

[0143] 1. Construction of the recombinant vector pBWA(V)BS-3HA-AtSRRM1L(9A)-GFP

[0144] 1) Obtaining the AtSRRM1L(9A) gene

[0145] Using the recombinant plasmid pET28a-AtSRRM1L(9A) as a template, PCR amplification was performed using the primer pair pBWA(V)BS-3HA-AtSRRM1L(9A)-GFP-SmaIF / R (SEQ ID NO.19 and SEQ ID NO.20) and the PrimeSTAR Max DNA Polymerase kit to obtain a PCR amplification product, which is the AtSRRM1L(9A) gene with SmaI restriction sites and homologous arms to the vector part.

[0146] 2) Construction of the recombinant vector pBWA(V)BS-3HA-AtSRRM1L(9A)

[0147] The pBWA(V)BS-3HA-GFP vector was digested with the restriction endonuclease SmaI. After the digestion products were recovered and purified by gel extraction, they were ligated with the above PCR products using the homologous recombination enzyme ClonExpress II One Step Cloning Kit to obtain the pBWA(V)BS-3HA-AtSRRM1L(9A)-GFP recombinant vector, and the pBWA(V)BS-3HA-AtSRRM1L(9A)-GFP recombinant vector was verified by sequencing.

[0148] 2. Construction of recombinant vectors pBWA(V)BS-3HA-AtSRRM1L(3A)-GFP and pBWA(V)BS-3HA-AtSRRM1L(6A)-GFP

[0149] 1) Obtaining of AtSRRM1L(3A) and AtSRRM1L(6A) genes

[0150] There are nine phosphorylation sites on the AtSRRM1L protein that can be recognized by AtSnRK1, located at Ser27, Thr41, Thr168, Ser285, Ser287, Ser391, Ser393, Ser41, and Ser558 respectively. Among them, the amino acids Ser27, Thr41, and Thr168 are located in the PWI conserved domain of the AtSRRM1L protein, and the amino acids Ser285, Ser287, Ser391, Ser393, Ser41, and Ser558 are located in the RS region of the AtSRRM1L protein. We replaced all the bases of the 3 phosphorylation sites in the PWI conserved domain of the AtSRRM1L protein with GCT, causing the amino acids at these 3 positions to all mutate into alanine (A). We re-synthesized the mutated AtSRRM1L gene and named it AtSRRM1L(3A) to simulate interrupted phosphorylation. Additionally, we replaced all the bases of the 6 phosphorylation sites in the RS region of the AtSRRM1L protein with GCT, causing the amino acids at these 6 positions to all mutate into alanine (A). We re-synthesized the mutated AtSRRM1L gene and named it AtSRRM1L(6A) to simulate interrupted phosphorylation.

[0151] Using the AtSRRM1L(3A) and AtSRRM1L(6A) genes as templates, PCR amplification was performed with the primer pairs pBWA(V)BS-3HA-AtSRRM1L(3A)-GFP-SmaIF / R (SEQ ID NO.19 and SEQ ID NO.20), pBWA(V)BS-3HA-AtSRRM1L(6A)-GFP-SmaIF / R (SEQ ID NO.19 and SEQ ID NO.20) and the PrimeSTAR Max DNA Polymerase kit to obtain PCR amplification products, namely the AtSRRM1L(3A) and AtSRRM1L(6A) genes with SmaI restriction sites and homologous arms to the vector part.

[0152] 2) Construction of recombinant vectors pBWA(V)BS-3HA-AtSRRM1L(3A)-GFP and pBWA(V)BS-3HA-AtSRRM1L(6A)-GFP

[0153] The pBWA(V)BS-3HA-GFP vector was digested with the restriction enzyme SmaI. After the digestion products were recovered and purified by gel extraction, they were ligated with the above PCR products respectively using the homologous recombination enzyme ClonExpress II One Step Cloning Kit to obtain the pBWA(V)BS-3HA-AtSRRM1L(3A)-GFP and pBWA(V)BS-3HA-AtSRRM1L(6A)-GFP recombinant vectors, and the above recombinant vectors were verified by sequencing.

[0154] 3. Transient transformation of tobacco

[0155] For subcellular localization analysis, the recombinant plasmids pBWA(V)BS-3HA-AtSRRM1L-GFP, pBWA(V)BS-3HA-AtSRRM1L(3A)-GFP, pBWA(V)BS-3HA-AtSRRM1L(6A)-GFP and pBWA(V)BS-3HA-AtSRRM1L(9A)-GFP were respectively transformed into Agrobacterium tumefaciens GV3101 and transiently expressed in tobacco leaves. After the tobacco was cultured for 2 days, the fluorescence signals of the interacting proteins were detected using a confocal laser microscope. The results are as Figure 7As shown in A of [reference], the full-length AtSRRM1L-GFP is localized to the nucleus in a typical speckled pattern. Both AtSRRM1L(6A) and AtSRRM1L(9A) lose the nuclear speckle characteristics and are evenly distributed in the nucleus, while the mutation of three phosphorylation sites on the PWI domain does not affect the nuclear speckle localization. These data indicate that the phosphorylation of the RS region of the AtSRRM1L protein mediated by AtSnRK1 is very important for nuclear speckle localization, and the RS region is the region that confers nuclear speckle localization to AtSRRM1L.

[0156] 4. Genetic transformation of Arabidopsis thaliana

[0157] 1) Transformation of Agrobacterium tumefaciens GV3101

[0158] The expression vectors pBWA(V)BS-3HA-GFP, pBWA(V)BS-3HA-AtSRRM1L-GFP, and pBWA(V)BS-3HA-AtSRRM1L(9A)-GFP were respectively transformed into Agrobacterium tumefaciens GV3101 using the freeze-thaw method. The specific operation steps are detailed in the Agrobacterium tumefaciens GV3101 competent cell instruction manual.

[0159] 2) Genetic transformation of Arabidopsis thaliana

[0160] Arabidopsis thaliana was genetically transformed using the Agrobacterium-mediated floral dip method. GFP, SRRM1L-GFP, and SRRM1L(9A)-GFP were overexpressed under the Col-0 background, and homozygous lines were obtained through screening. T3-generation seeds were disinfected and sown on 1 / 2 MS medium containing 25 mg / L Basta resistance, and subcellular localization was performed on the root tip cells of 5-day-old seedlings. The results are as Figure 7 shown in B of [reference]. Under normal conditions, SRRM1L-GFP is localized to the nuclear speckles in the root tip cells of Arabidopsis thaliana seedlings, which is consistent with the localization observed in tobacco leaf cells. Subsequently, the SRRM1L-GFP transgenic seedlings were treated with salt. The results showed that compared with the control, after salt treatment, SRRM1L-GFP was present in more nuclear speckles. Whether or not salt treatment was used, the cells of SRRM1L(9A)-GFP transgenic seedlings showed that GFP was evenly distributed in the nucleus, and no nuclear speckles were found. These results indicate that the increase in nuclear speckles of SRRM1L in response to salt stress is promoted through the SnRK1-SRRM1L pathway, and this promotion is partially dependent on SnRK-mediated phosphorylation.

[0161] Example 6: Phosphorylation of AtSRRM1L by AtSnRK1 affects the alternative splicing of the precursor mRNA of the target gene NFYA10

[0162] 1. Construction of recombinant vector pET28a-AtSRRM1L(9D)

[0163] 1) Acquisition of the AtSRRM1L(9D) gene

[0164] It is known that there are nine phosphorylation sites on the AtSRRM1L protein that can be recognized by AtSnRK1, namely Ser27, Thr41, Thr168, Ser285, Ser287, Ser391, Ser393, Ser41 and Ser558. Therefore, we mutated the serine (S) at these nine positions of the AtSRRM1L protein to aspartic acid (D) and the threonine (T) to glutamic acid (E). We resynthesized the mutated AtSRRM1L gene and named it AtSRRM1L(9D). The AtSRRM1L(9D) protein encoded by the AtSRRM1L(9D) gene has the ability to be phosphorylated by the AtSnRK1 protein.

[0165] Using the AtSRRM1L (9D) gene as a template, PCR amplification was performed using primers pET28a-HA-AtSRRM1L (9D)-SalIF / R (SEQ ID NO. 17 and SEQ ID NO. 18) and a PrimeSTARMax DNA Polymerase kit to obtain a PCR amplification product, namely the AtSRRM1L (9D) gene with a SalI restriction site and partial homology arms with the vector.

[0166] 2) Construction of the recombinant vector pET28a-AtSRRM1L(9D)

[0167] The pET28a vector was digested with the restriction endonuclease SalI. The digested product was purified by gel recovery and then ligated with the PCR product using the homologous recombinase ClonExpress II One Step Cloning Kit to obtain the pET28a-AtSRRM1L (9D) recombinant vector, which was then sequenced and verified.

[0168] 2. Protein expression and purification

[0169] Transform the protein expression vector pET28a-AtSRRM1L(9D) into competent E. coli BL21(DE3). For detailed procedures, refer to the BL21(DE3) Chemically Competent Cell instructions. Obtain BL21(DE3) E. coli containing the pET28a-AtSRRM1L(9D) protein expression vector and induce protein expression. Purify the expressed AtSRRM1L(9D) protein using the Kangwei Century His-Tagged Protein Purification Kit.

[0170] 3. In vitro REMSA analysis

[0171] To examine whether AtSnRK1-mediated phosphorylation affects the RNA binding activity of AtSRRM1L, in vitro REMSA analysis was performed. Figure 8 As shown in Figure 5A, the mRNA binding ability of phosphorylated SRRM1L (9D) was higher than that of SRRM1L (wt), indicating that AtSnRK1-mediated phosphorylation of AtSRRM1L promoted the ability of AtSRRM1L to bind to target mRNA.

[0172] 4. In planta RIP-PCR analysis

[0173] GFP or AtSRRM1-GFP genes were overexpressed in wild-type (Col-0) or 2kinm backgrounds, and the ability of GFP or AtSRRM1L to bind to RNA in overexpressing plants GFP / Col-0, AtSRRM1-GFP / Col-0, GFP / 2kinm, and AtSRRM1-GFP / 2kinm was determined by RIP-PCR. Figure 8 As shown in Figure 3B, GFP and AtSRRM1L-GFP proteins were immunoprecipitated from GFP / Col-0, AtSRRM1-GFP / Col-0, GFP / 2kinm, and AtSRRM1-GFP / 2kinm plants using GFP antibodies, respectively. In AtSRRM1-GFP / Col-0 overexpressing plants, AtSRRM1L binding to the first exon of NFYA10 pre-mRNA was more enriched than in AtSRRM1-GFP / 2kinm overexpressing plants, indicating that AtSnRK1 phosphorylation can enhance the binding of AtSRRM1L to the target RNA.

[0174] Example 7: Genetic transformation of AtSRRM1L and expression analysis in transgenic Arabidopsis

[0175] 1. Recombinant vector pCAMBIA3301-Pro SRRM1L-GFP, pCAMBIA3301-Pro SRRM1L -AtSRRM1L-GFP, pCAMBIA3301-Pro SRRM1L -AtSRRM1L(9A)-GFP and pCAMBIA3301-Pro SRRM1L -Construction of AtSRRM1L(9D)-GFP

[0176] 1) Obtaining the promoter sequence of the AtSRRM1l gene

[0177] Using the genomic DNA of wild-type Arabidopsis thaliana as a template, and performing PCR amplification with the primer pair pCAMBIA3301-Pro SRRM1L -GUS-EcoRINcoIF / R (SEQ ID NO.24 and SEQ ID NO.25) and the PrimeSTAR Max DNA Polymerase kit to obtain a PCR amplification product, that is, the Pro promoter with EcoRINcoI restriction enzyme sites and homologous arms with the vector part SRRM1L promoter.

[0178] Double-digesting the pCAMBIA3301 vector with the restriction enzymes EcoRI and NcoI. After the digestion products are recovered and purified by gel electrophoresis, they are ligated with the above PCR product using the homologous recombination enzyme ClonExpress II One Step Cloning Kit to obtain the pCAMBIA3301-Pro SRRM1L -GUS recombinant vector, and sequencing and verifying the pCAMBIA3301-Pro SRRM1L -GUS recombinant vector.

[0179] 2) Construction of the recombinant vector pBWA(V)BS-3HA-AtSRRM1L(9D)-GFP

[0180] Using the recombinant plasmid PET28a-AtSRRM1L(9D) as a template, and performing PCR amplification with the primer pair pBWA(V)BS-3HA-AtSRRM1L(9D)-GFP-SmaIF / R (SEQ ID NO.19 and SEQ ID NO.20) and the PrimeSTAR Max DNA Polymerase kit to obtain a PCR amplification product, that is, the AtSRRM1L(9D) gene with SmaI restriction enzyme sites and homologous arms with the vector part

[0181] The pBWA(V)BS-3HA-GFP vector was digested with the restriction endonuclease SmaI. After the digestion products were purified by gel extraction, they were ligated with the above PCR products using the homologous recombination enzyme ClonExpress II One Step Cloning Kit to obtain the pBWA(V)BS-3HA-AtSRRM1L(9D)-GFP recombinant vector, and the pBWA(V)BS-3HA-AtSRRM1L(9D)-GFP recombinant vector was verified by sequencing.

[0182] 3) Construction of the recombinant vector pCAMBIA3301-Pro SRRM1L -GFP, pCAMBIA3301-Pro SRRM1L -AtSRRM1L-GFP, pCAMBIA3301-Pro SRRM1L -AtSRRM1L(9A)-GFP and pCAMBIA3301-Pro SRRM1L -Construction of AtSRRM1L(9D)-GFP

[0183] Using the recombinant plasmids pBWA(V)BS-3HA-GFP, pBWA(V)BS-3HA-AtSRRM1L-GFP, pBWA(V)BS-3HA-AtSRRM1L(9A)-GFP and pBWA(V)BS-3HA-AtSRRM1L(9D)-GFP as templates respectively, and using the primer pairs pCAMBIA3301-Pro SRRM1L -GFP-NcoIPmlIF / R (SEQ ID NO.26 and SEQ ID NO.27), the primer pairs pCAMBIA3301-Pro SRRM1L -AtSRRM1L-GFP-NcoIPmlIF / R (SEQ ID NO.28 and SEQ ID NO.29), the primer pairs pCAMBIA3301-Pro SRRM1L -AtSRRM1L(9A)-GFP-NcoIPmlIF / R (SEQ ID NO.28 and SEQ IDNO.29), the primer pairs pCAMBIA3301-Pro SRRM1L -AtSRRM1L(9D)-GFP-NcoIPmlIF / R (SEQ ID NO.28 and SEQ ID NO.29) and the PrimeSTAR Max DNA Polymerase kit for PCR amplification to obtain PCR amplification products, namely the GFP gene, AtSRRM1L-GFP gene, AtSRRM1L(9A)-GFP and AtSRRM1L(9D)-GFP genes with NcoIPmlI restriction sites and homologous arms to the vector part.

[0184] The pCAMBIA3301-Pro SRRM1L -GUS vector was digested with the restriction endonucleases NcoI and PmlI. After the digestion products were recovered and purified by gel extraction, they were ligated with the above PCR products respectively using the homologous recombinase ClonExpress II One Step Cloning Kit to obtain pCAMBIA3301-Pro SRRM1L -GFP, pCAMBIA3301-Pro SRRM1L -AtSRRM1L-GFP, pCAMBIA3301-Pro SRRM1L -AtSRRM1L(9A)-GFP and pCAMBIA3301-Pro SRRM1L -AtSRRM1L(9D)-GFP recombinant vectors, and the above recombinant vectors were verified by sequencing.

[0185] 2. Construction of the recombinant vectors pCAMBIA1302-Pro SRRM1L -AtSRRM1L, pCAMBIA1302-Pro SRRM1L -AtSRRM1L(9A) and pCAMBIA1302-Pro SRRM1L -AtSRRM1L(9D)

[0186] Using the recombinant plasmids pCAMBIA3301-Pro SRRM1L -AtSRRM1L-GFP, pCAMBIA3301-Pro SRRM1L -AtSRRM1L(9A)-GFP and pCAMBIA3301-Pro SRRM1L -AtSRRM1L(9D)-GFP as templates respectively, PCR amplifications were carried out using the primer pairs pCAMBIA1302-Pro SRRM1L -AtSRRM1L-KpnI NcoIF / R (SEQ ID NO.30 and SEQ ID NO.31), the primer pairs pCAMBIA1302-Pro SRRM1L -AtSRRM1L(9A)-KpnI NcoIF / R (SEQ ID NO.30 and SEQ ID NO.31), the primer pairs pCAMBIA1302-Pro SRRM1L -AtSRRM1L(9D)-KpnI NcoIF / R (SEQ ID NO.30 and SEQ ID NO.31) and the PrimeSTAR Max DNA Polymerase kit to obtain PCR amplification products, namely Pro with KpnI NcoI restriction sites and homologous arms with the vector partSRRM1L -AtSRRM1L gene, Pro SRRM1L -AtSRRM1L(9A) gene and Pro SRRM1L -AtSRRM1L(9D) gene.

[0187] The pCAMBIA1302 vector was double-digested with restriction enzymes KpnI and NcoI. After the digestion products were recovered and purified by gel extraction, they were ligated with the above PCR products respectively using the homologous recombination enzyme ClonExpress II One Step Cloning Kit to obtain pCAMBIA1302-Pro SRRM1L -AtSRRM1L, pCAMBIA1302-Pro SRRM1L -AtSRRM1L(9A) and pCAMBIA1302-Pro SRRM1L -AtSRRM1L(9D) recombinant vectors, and the above recombinant vectors were verified by sequencing.

[0188] 3. Recombinant vector pCAMBIA1302-Pro SnRK1 -AtSnRK1 and pCAMBIA1302-Pro SnRK1 -Construction of AtSnRK1(K48M)

[0189] 1) Construction of recombinant vector pCAMBIA1302-Pro SnRK1 of

[0190] Using wild-type Arabidopsis genomic DNA as a template, PCR amplification was carried out with the primer pair pCAMBIA1302-Pro SnRK1 -KpnINcoIF / R (SEQ ID NO.32 and SEQ ID NO.33) and PrimeSTAR Max DNA Polymerase kit to obtain a PCR amplification product, that is, Pro SnRK1 promoter with EcoRI and NcoI restriction sites and homologous arms with the vector part.

[0191] The pCAMBIA1302 vector was double-digested with restriction enzymes KpnI and NcoI. After the digestion products were recovered and purified by gel extraction, they were ligated with the above PCR products using the homologous recombination enzyme ClonExpress II One Step Cloning Kit to obtain pCAMBIA1302-Pro SnRK1 recombinant vector, and the pCAMBIA1302-Pro SnRK1 recombinant vector was verified by sequencing.

[0192] 2) Recombinant vector pCAMBIA1302-ProSnRK1 -AtSnRK1 and pCAMBIA1302-Pro SnRK1 -Construction of AtSnRK1(K48M)

[0193] Using the recombinant plasmids pGBKT7-AtSnRK1 and PET28a-AtSnRK1(K48M) as templates, and the primer pairs pCAMBIA1302-Pro SnRK1 -AtSnRK1-NcoIF / R (SEQ ID NO.34 and SEQ ID NO.35), primer pairs pCAMBIA1302-Pro SnRK1 -AtSnRK1(K48M)-NcoIF / R (SEQ ID NO.34 and SEQ ID NO.35) and PrimeSTAR Max DNA Polymerase kit for PCR amplification to obtain the PCR amplification products, namely Pro SnRK1 -AtSnRK1 gene and Pro SnRK1 -AtSnRK1(K48M) gene.

[0194] Digest the pCAMBIA1302-Pro SnRK1 vector with the restriction endonuclease NcoI. After the digestion products are recovered and purified by gel extraction, use the homologous recombination enzyme ClonExpress II One Step Cloning Kit to ligate with the above PCR products respectively to obtain pCAMBIA1302-Pro SnRK1 -AtSnRK1 and pCAMBIA1302-Pro SnRK1 -AtSnRK1(K48M) recombinant vectors, and sequence verification is carried out on the above recombinant vectors.

[0195] 4. Transformation of Agrobacterium tumefaciens GV3101

[0196] Using the freeze-thaw method, the expression vectors pBWA(V)BS-3HA-GFP, pBWA(V)BS-3HA-AtSRRM1L-GFP, pBWA(V)BS-3HA-AtSRRM1L(9A)-GFP, pCAMBIA3301-Pro SRRM1L -GFP, pCAMBIA3301-Pro SRRM1L -AtSRRM1L-GFP, pCAMBIA3301-Pro SRRM1L -AtSRRM1L(9A)-GFP, pCAMBIA1302-Pro SRRM1L -AtSRRM1L, pCAMBIA1302-ProSRRM1L -AtSRRM1L(9A), pCAMBIA1302-Pro SRRM1L -AtSRRM1L(9D), pCAMBIA1302-Pro SnRK1 -AtSnRK1 and pCAMBIA1302-Pro SnRK1 -AtSnRK1(K48M) were transformed into Agrobacterium tumefaciens GV3101, and the specific operation steps are detailed in Weidi Agrobacterium tumefaciens GV3101 competence instructions.

[0197] 5. Genetic transformation of transgenic Arabidopsis thaliana mediated by Agrobacterium tumefaciens GV3101 and phenotypic analysis of plants under salt stress

[0198] Arabidopsis thaliana was genetically transformed using the Agrobacterium-mediated floral dip method. Homozygous lines were obtained by overexpressing GFP, SRRM1L-GFP, and SRRM1L(9A)-GFP in the Col-0 background or complementing GFP in the srrm1l mutant background. Figure 9 As shown in A, on 1 / 2MS medium, compared with the normally complemented SRRM1-GFP line, the expression of the SRRM1L (9A) gene with a phosphorylation site mutation could not rescue the salt-sensitive phenotype of the srrm1l mutant. After salt stress treatment, the SRRM1L (9A)-GFP complemented line showed shorter root length and lower survival rate compared with the SRRM1L-GFP complemented line. In addition, under normal conditions, there was no significant difference in root length between the SRRM1L (9A)-GFP overexpression line and the SRRM1L-GFP overexpression line, but under salt stress treatment, the root length of the SRRM1L (9A)-GFP overexpression line seedlings was significantly lower than that of the SRRM1L-GFP overexpression line, and the seedling survival rate was also relatively low. The salt tolerance of plants of different genotypes in soil was also determined, and the results are shown in Figure 2. Figure 9 As shown in Figure B, consistent with the results of the seedling stage, under normal growth conditions, there were no significant differences in the survival rate and relative chlorophyll content of different genotypes. Although salt stress reduced the survival rate of plants of different genotypes, the survival rate of SRRM1L-GFP overexpressing plants was higher than that of other genotypes, while SRRM1L(9A)-GFP overexpressing plants were more sensitive to salt.

[0199] To further explore the relationship between SnRK1 and SRRM1L, the loss-of-function mutant 2kinm was crossed with the srrm1l mutant to generate the 2kinm / srrm1l double mutant. A series of complementation lines were performed in the double mutant background. First, the SRRM1L and SnRK1 genes were simultaneously introduced into the 2kinm / srrm1l double mutant to generate independent complementation lines. Figure 9As shown in C in [reference], the complementation of the SRRM1L and SnRK1 genes completely rescued the salt-sensitive response of the 2kinm / srrml1 mutant. When the SRRM1L(9A) and SnRK1(K48M) genes were simultaneously complemented into the 2kinm / srrm1l double mutant, it was found that the SnRK1(K48M) / SRRM1L(9A) complementation line had the same phenotype as the 2kinm / srrm1l double mutant and was highly sensitive to salt. When comparing the phosphorylation site mutations of SRRM1L, it was found that the SnRK1 / SRRM1L(9A) complementation line was highly sensitive to salt, while the SnRK1 / SRRM1L(9D) complementation line had higher salt tolerance than the SnRK1 / SRRM1L complementation line. In addition, the salt tolerances of some other complementation lines, such as SnRK1(K48M) / SRRM1L and SnRK1(K48M) / SRRM1L(9D), were lower than that of the SnRK1 / SRRM1L complementation line. Next, the survival rates and chlorophyll contents of plants with different genotypes under control or salt treatment were evaluated, and similar conclusions were drawn as those of the inhibited plant growth after salt treatment. The above results indicate that the phosphorylation of SRRM1L by SnRK1 is crucial for the role of SRRM1L in salt tolerance.

[0200] Example 8: Genetic transformation of AtSRRM1L and expression analysis in transgenic soybeans

[0201] I. Construction of the pCAMBIA3301-RNAi-GmSRRM1L.1 / .2 expression vector

[0202] The non-conserved regions of the GmSRRM1L.1 (2343-2573bp) and GmSRRM1L.2 (2274-2504bp) genes were selected. Since both the GmSRRM1L.1 and GmSRRM1L.2 genes are homologous genes of AtSRRM1L and have a high similarity, a same and non-conserved sequence needs to be selected as the target sequence for RNAi technology, that is, two soybean endogenous genes are knocked out by RNAi technology.

[0203] 1. Construction of the pHANNIBAL-RNAi-GmSRRM1L.1 / .2 forward recombinant vector

[0204] After the selected target sequence was artificially synthesized, PCR amplification was performed using the primer pair pHANNIBAL-RNAi-GmSRRM1L.1 / .2-XhoIF / R (SEQ ID NO.36 and SEQ ID NO.37) and the PrimeSTARMax DNA Polymerase kit to obtain a PCR amplification product, that is, the GmSRRM1L.1 / .2 gene fragment with XhoI restriction enzyme sites and homologous arms to the vector part.

[0205] The pHANNIBAL vector was digested with the restriction endonuclease XhoI, and then a part of GmSRRM1L.1 / .2 was ligated to the digested and purified vector using a homologous recombinase, while keeping the other sequences of the pHANNIBAL vector unchanged, to obtain the pHANNIBAL-RNAi-GmSRRM1L.1 / .2 forward recombinant vector. The pHANNIBAL-RNAi-GmSRRM1L.1 / .2 forward recombinant vector was verified by sequencing.

[0206] 2. Construction of the pHANNIBAL-RNAi-GmSRRM1L.1 / .2 vector

[0207] After the antisense fragment of the selected target sequence was artificially synthesized, PCR amplification was performed using the primer pair pHANNIBAL-RNAi-GmSRRM1L.1 / .2-XbaIF / R (SEQ ID NO.38 and SEQ ID NO.39) and the PrimeSTAR Max DNA Polymerase kit to obtain a PCR amplification product, which is the GmSRRM1L.1 / .2 antisense fragment with an XbaI restriction site and a partial homologous arm to the vector.

[0208] The above pHANNIBAL-RNAi-GmSRRM1L.1 / .2 forward vector was digested with the restriction endonuclease XbaI, and then a part of the GmSRRM1L.1 / .2 antisense fragment was ligated to the digested and purified vector using a homologous recombinase, while keeping the other sequences of the pHANNIBAL-RNAi-GmSRRM1L.1 / .2 forward vector unchanged, to obtain the pHANNIBAL-RNAi-GmSRRM1L.1 / .2 recombinant vector. The pHANNIBAL-RNAi-GmSRRM1L.1 / .2 recombinant vector was verified by sequencing.

[0209] The sequencing results showed that the pHANNIBAL-RNAi-GmSRRM1L.1 / .2 recombinant vector was obtained by digesting the pHANNIBAL-RNAi-GmSRRM1L.1 / .2 forward vector with XbaI and inserting a part of the GmSRRM1L.1 / .2 antisense fragment, while keeping the other sequences of the pHANNIBAL-RNAi-GmSRRM1L.1 / .2 forward vector unchanged.

[0210] 3. Construction of the pCAMBIA3301-RNAi-GmSRRM1L.1 / .2 expression vector

[0211] Using the above pHANNIBAL-RNAi-GmSRRM1L.1 / .2 plasmid as a template, PCR amplification was performed with the primer pair 3301-RNAi-GmSRRM1L.1 / .2-EcoRI NcoIF / R (SEQ ID NO.40 and SEQ ID NO.41) and the PrimeSTAR Max DNA Polymerase kit to obtain the PCR amplification products of the CaMV 35S promoter, partial sense fragment, intron, and partial antisense fragment.

[0212] The pCAMBIA3301 vector was double digested with the restriction enzymes EcoRI and NcoI, and then the CaMV 35S promoter, partial sense fragment, intron, and partial antisense fragment were ligated to the digested and purified vector using homologous recombinase to obtain the pCAMBIA3301-RNAi-GmSRRM1L.1 / .2 recombinant vector, and the pCAMBIA3301-RNAi-GmSRRM1L.1 / .2 recombinant vector was verified by sequencing.

[0213] The sequencing results showed that the pCAMBIA3301-RNAi-GmSRRM1L.1 / .2 recombinant vector was obtained by replacing the DNA fragment between the EcoRI and NcoI restriction sites of the pCAMBIA3301 vector with the CaMV 35S promoter, partial sense fragment, intron, and partial antisense fragment genes, while keeping the other sequences of the pCAMBIA3301 vector unchanged.

[0214] II. Transformation of Agrobacterium tumefaciens EHA105

[0215] The expression vectors pBWA(V)BS-3HA-GFP, pBWA(V)BS-3HA-AtSRRM1L-GFP, pBWA(V)BS-3HA-AtSRRM1L(9A)-GFP, and pCAMBIA3301-RNAi-GmSRRM1L.1 / .2 were respectively transformed into Agrobacterium tumefaciens EHA105 by the freeze-thaw method. The specific operation steps are detailed in the instruction manual of the competent cells of Agrobacterium rhizogenes K599. Instruction Manual of Competent Cells of Agrobacterium rhizogenes K599.

[0216] III. Genetic Transformation of Transgenic Soybeans Mediated by Agrobacterium tumefaciens EHA105 and Phenotypic Analysis of Plants under Salt Stress

[0217] 1. Obtaining Transgenic Soybeans

[0218] 1) Induction: Select healthy, mature soybean seeds and sterilize them using chlorine gas sterilization (100 mL of sodium hypochlorite (8% effective concentration) in 5 mL of concentrated hydrochloric acid) for 16-20 hours. This procedure is performed in a fume hood. After sterilization, the seeds are air-blown for 24 hours in a clean bench to dissipate any residual chlorine. Seal the petri dish containing the sterilized soybeans and store at 4°C until ready for use.

[0219] 2) Pre-culture: Inoculate the sterilized seeds with the hilum facing downward on the germination medium, place in a 25°C incubator, and culture in the dark for 1 day.

[0220] 3) Preparation of Agrobacterium culture: Agrobacterium carrying the target gene plasmid is plated onto the appropriate culture medium for initial activation. After 48 hours of culture, the cells are harvested and reactivated on fresh culture medium. After 24 hours of culture, the cells are harvested and placed in the infection solution. Vortex the mixture and adjust the culture solution to an OD value of 0.5 using a spectrophotometer.

[0221] 4) Infection: Wound the germinated soybeans and then add the prepared bacterial solution to complete the infection. Discard the infection solution and place the explants on a co-cultivation medium lined with filter paper. Co-cultivate at 25°C in the dark for 3-5 days.

[0222] 5) Recovery culture: Select explants that have grown well and are free of contamination after co-culture, cut off the end of the embryonic axis, insert them into the recovery solid culture medium, and resume culture for 7-10 days.

[0223] 6) Screening culture: The explants that have grown clustered shoots after recovery culture are inoculated onto screening culture medium and cultured under 16h / 8h light / dark conditions for 21 days.

[0224] 7) Elongation culture: The well-growing clustered shoots after screening were transferred to a new elongation medium and cultured under 16h / 8h light / dark conditions for 21 days of screening culture.

[0225] 8) Rooting culture: When the shoots grow to about 5 cm, transfer them to rooting medium for further screening under a 16 h / 8 h light / dark cycle for 21 days.

[0226] 9) Positive seedling detection: The presence of bar protein in transgenic plants was directly identified using the Bar test paper method.

[0227] 10) Hardening: Remove seedlings that have tested positive with the Bar test strips from the culture medium. Wash any culture medium attached to the roots and transfer them to seedling trays filled with nutrient soil. Incubate at 27°C with a 16 / 8 hour light / dark cycle for 3-4 weeks.

[0228] 2. Identification of genetically modified soybeans

[0229] 1) Identification of pBWA(V)BS-3HA-GFP, pBWA(V)BS-3HA-AtSRRM1L-GFP and pBWA(V)BS-3HA-AtSRRM1L(9A)-GFP transgenic soybeans

[0230] The Bar resistance gene in transgenic soybeans was detected by PCR. Randomly select 2 - 3 leaves of transgenic soybeans, put them into a centrifuge tube and add 35 μL of Lysis Buffer A. Heat at 95 °C for 10 min. After standing, take 1 μL of the supernatant as the template for the PCR reaction system. PCR amplification was carried out using the primer pair Bar-F / R (SEQ ID NO.42 and SEQ ID NO.43) and the PrimeSTAR Max DNA Polymerase kit. The resistance gene fragments carried by the pBWA(V)BS-3HA-GFP, pBWA(V)BS-3HA-AtSRRM1L-GFP and pBWA(V)BS-3HA-AtSRRM1L(9A)-GFP vectors were detected by PCR to obtain PCR amplification products. The Bar gene was cloned, indicating that the relevant target genes had been expressed in transgenic soybeans.

[0231] 2) Identification of pCAMBIA3301-RNAi-GmSRRM1L.1 / .2 transgenic soybeans

[0232] The transgenic soybeans were identified by PCR. Randomly select 2 - 3 leaves of transgenic soybeans, put them into a centrifuge tube and add 35 μL of Lysis Buffer A. Heat at 95 °C for 10 min. After standing, take 1 μL of the supernatant as the template for the PCR reaction system. PCR amplification was carried out using the primer pair PDK-F / R (SEQ ID NO.44 and SEQ ID NO.45) and the PrimeSTAR Max DNA Polymerase kit. The specific gene fragments carried by the pCAMBIA3301-RNAi-GmSRRM1L.1 / .2 vector were detected by PCR to obtain PCR amplification products. The PDK gene was cloned, indicating that the relevant target genes had been expressed in transgenic soybeans.

[0233] 3) Western blot detection of the expression of SRRM1L protein in transgenic soybeans.

[0234] Extract the total proteins of transgenic soybean plants of pBWA(V)BS-3HA-GFP, pBWA(V)BS-3HA-AtSRRM1L-GFP, pBWA(V)BS-3HA-AtSRRM1L(9A)-GFP and pCAMBIA3301-RNAi-GmSRRM1L.1 / .2, and use the customized antibody of SRRM1L to detect the expression of SRRM1L protein in transgenic soybean plants.

[0235] The results are as Figure 10 shown in A of the figure. The Western blot results indicate that AtSRRM1L and AtSRRM1L(9A) proteins have been successfully expressed in pBWA(V)BS-3HA-AtSRRM1L-GFP and pBWA(V)BS-3HA-AtSRRM1L(9A)-GFP transgenic soybean plants. The GmSRRM1L.1 and GmSRRM1L.2 genes in pCAMBIA3301-RNAi-GmSRRM1L.1 / .2 transgenic soybean plants have been successfully knocked out, and no GmSRRM1L.1 and GmSRRM1L.2 proteins are detected.

[0236] 3. Analysis of the phenotypes and physiological indexes of transgenic soybean plants under salt stress

[0237] Mix the soil according to the ratio of peat soil: vermiculite: perlite of 3:1:1 and soak it thoroughly, then put it into the planting pots, leaving a gap of 2 - 3 cm high; select healthy and mature transgenic soybean seeds, evenly put them into the planting pots, and then cover with about 1 cm of nutrient soil and place them under 16 h of light for cultivation. When the seedlings grow to 4 weeks old, conduct stress treatment on them. For salt stress treatment, irrigate the seedlings that have been normally cultivated for 4 weeks in the pots with Hoagland's nutrient solution containing 200 mM NaCl for 10 days, and change the culture solution regularly during this period. Analyze the phenotypes and related physiological data of transgenic soybean plants. By counting physiological indexes such as the content of malondialdehyde and chlorophyll, all experimental techniques are repeated and biologically replicated 3 times each.

[0238] The results are as Figure 10 shown in B of the figure. Under normal conditions, the growth states of each group of plants are similar. After salt treatment, the growth of wild-type plants and overexpression empty vector plants is inhibited, showing yellowing leaves. The growth of RNAi-GmSRRM1L.1 / .2 transgenic soybean plants is severely affected, showing severe leaf whitening and even withering. The transgenic soybean plants of pBWA(V)BS-3HA-AtSRRM1L-GFP have the best growth state, followed by the transgenic soybean plants of pBWA(V)BS-3HA-AtSRRM1L(9A)-GFP, indicating that the phosphorylation of SRRM1L by SnRK1 is crucial for plant salt tolerance.

[0239] As Figure 10 shown by C in [reference], under normal conditions, there are no significant differences in physiological indexes such as malondialdehyde and chlorophyll contents among each group of plants. However, after salt treatment, the malondialdehyde contents of wild-type plants, pBWA(V)BS-3HA-GFP, pBWA(V)BS-3HA-AtSRRM1L(9A)-GFP, and pCAMBIA3301-RNAi-GmSRRM1L.1 / .2 transgenic soybeans are all significantly increased compared with those under normal conditions, and the chlorophyll contents are all decreased compared with those under normal conditions. The pBWA(V)BS-3HA-AtSRRM1L-GFP transgenic soybean plants grow best. This indicates that AtSRRM1L is crucial for improving plant salt tolerance.

[0240] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. Application of protein AtSRRM1L in improving plant salt tolerance, characterized in that: The protein AtSRRM1L is any one of the following proteins a) and b): a) a protein with an amino acid sequence as shown in SEQ ID NO.1; b) a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein with the amino acid sequence as shown in SEQ ID NO. 1; The plant is Arabidopsis thaliana or soybean.

2. The use according to claim 1, characterized in that The application is to overexpress the protein AtSRRM1L or co-express the proteins AtSRRM1L and AtSnRK1 in plants.

3. Use of the gene encoding the protein AtSRRM1L according to claim 1 in improving plant salt tolerance, characterized in that: The nucleotide sequence of the encoding gene is shown in SEQ ID NO. 2; the plant is Arabidopsis thaliana or soybean.

4. Use of a recombinant vector containing the coding gene according to claim 3 in improving plant salt tolerance, characterized in that: The plant is Arabidopsis thaliana or soybean.

5. Use of a recombinant bacterium containing the coding gene according to claim 3 or the recombinant vector according to claim 4 in improving plant salt tolerance, characterized in that: The plant is Arabidopsis thaliana or soybean.

6. A method for cultivating transgenic soybeans with salt tolerance, characterized in that: The gene encoding the protein AtSRRM1L according to claim 1 is introduced into soybean, and the nucleotide sequence of the gene encoding the protein AtSRRM1L is shown as SEQ ID NO.

2.

7. The method according to claim 6, characterized in that The transgenic soybean is a transgenic soybean obtained by induction with Agrobacterium tumefaciens EHA105.

Images