Cold-resistant gene TrWRKY10a540 as well as expression vector, application and breeding method thereof

Abstract

The invention provides a cold-resistant gene TrWRKY10a540 as well as an expression vector, application and a breeding method thereof, and belongs to the field of plant molecular inheritance and genetic improvement. In particular discloses a cold-resistant gene TrWRKY10a540 derived from Trifolium repens L., an expression vector and application of the cold-resistant gene TrWRKY10a540 to improvement of cold resistance of plants, and solves the problem of insufficient research on low-temperature stress related genes of Trifolium repens L. in the prior art. The invention further discloses a breeding method for regulating and controlling the cold resistance of plants through the cold-resistant gene TrWRKY10a540, and research results show that the gene can remarkably improve the cold resistance and the antioxidant level of the plants under the low-temperature stress condition and has a good functional effect. The gene can be widely applied to plant molecular breeding, is particularly suitable for improving the low-temperature resistance of economic crops such as pasture, provides a new genetic resource for breeding of cold-resistant plants, and has important significance for revealing a regulation mechanism of the cold resistance of the plants.

Description

Technical Field

[0001] The present invention belongs to the field of plant molecular genetics and genetic improvement, and particularly relates to a WRKY transcription factor gene TrWRKY10a540 derived from white clover and its application in improving plant cold resistance. Background Art

[0002] White Clover ( Trifolium repens White clover (L. repens), a perennial legume widely distributed in temperate and cool temperate regions, is renowned for its high yield, high quality, and strong adaptability. However, it is highly susceptible to abiotic stresses during its growth, particularly during the harsh winters of high latitudes. This often leads to abnormal mortality, severely restricting its production and promotion.

[0003] Plant cold tolerance is regulated by a variety of transcription factors, among which WRKY family members have attracted widespread attention for their functions in regulating stress response pathways and enhancing antioxidant systems. However, WRKY family members from the forage grass white clover have not been comprehensively studied, making further exploration of the cold tolerance mechanisms of WRKY family members particularly important. Summary of the Invention

[0004] The present invention aims to address the technical gap in the existing technology regarding the research on genes related to low temperature stress of white clover (Trifolium repens L.), and provides a cold-resistant gene TrWRKY10a540 derived from white clover and its application in improving plant cold resistance.

[0005] A gene that improves plant cold resistance, characterized in that the gene is the white clover TrWRKY10a540 gene.

[0006] Furthermore, the nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0007] Furthermore, the amino acid sequence encoded by the gene is shown as SEQ ID NO.2.

[0008] Application of TrWRKY10a540 gene in plant resistance to cold stress.

[0009] An expression vector for improving plant cold resistance is disclosed. The expression vector is a pCAMBIA1300-TrWRKY10a540 expression vector containing the TrWRKY10a540 gene.

[0010] Application of pCAMBIA1300-TrWRKY10a540 expression vector in plant resistance to cold stress.

[0011] A breeding method for regulating plant resistance to cold stress, comprising the following steps: S1: The coding sequence of TrWRKY10a540 from white clover was cloned using primers to obtain the gene clone sequence; S2: Connect the gene clone sequence to the vector pCAMBIA1300 to obtain the expression vector; S3: Introduce the expression vector obtained in S2 into Agrobacterium to obtain recombinant Agrobacterium; S4: Infect plants with the recombinant Agrobacterium obtained in S3 to obtain transgenic plants.

[0012] Furthermore, the Agrobacterium described in S3 is GV3101.

[0013] Furthermore, the plant described in S4 is Arabidopsis thaliana.

[0014] Furthermore, the infection in S4 is an inflorescence infection method.

[0015] Beneficial effects These experiments confirmed that the TrWRKY10a540 gene enhances plant cold tolerance and antioxidant capacity under low-temperature stress. Arabidopsis plants overexpressing this gene exhibited improved growth after low-temperature treatment, with significantly increased superoxide dismutase (SOD) and catalase (CAT) activities. These results suggest that the TrWRKY10a540 gene has important application value in plant cold tolerance breeding and could serve as an excellent cold-stress response gene resource for widespread use in cold-resistance research in forages and other economic crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the PCR identification image of the TrWRKY10a540 gene positive colony; Figure 2 This is the predicted domain structure of TrWRKY10a540 protein; Figure 3 This is the T3 generation Arabidopsis PCR positive screening image; Figure 4 The phenotypes of transgenic and wild-type Arabidopsis after low temperature stress; Figure 5 This is a diagram showing the expression analysis of the TrWRKY10a540 gene in transgenic plants; Figure 6 Figure 3 is the test result of physiological and biochemical indicators of transgenic Arabidopsis thaliana under low temperature stress, where A is chlorophyll (CHL) content, B is malondialdehyde (MDA) content, C is proline (Pro) content, D is catalase (CAT) content, E is peroxidase (POD) content, and F is superoxide dismutase (SOD) content. DETAILED DESCRIPTION

[0017] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is obvious that relevant persons can modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present invention to implement and apply the technology of the present invention.

[0018] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments. The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the materials, reagents, methods and instruments used are all conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained from commercial channels by those skilled in the art.

[0019] The primers described in the following examples were synthesized by Shanghai Sangon Biotechnology Co., Ltd.; sequencing was performed by Shanghai Sangon Biotechnology Co., Ltd.; restriction endonucleases, ligases, pMD™18-T Vector Cloning Kit, DNA Marker, PrimeSTAR Max Premix (2X), and reverse transcription kits used in the experiments were purchased from Takara; DH5α competent cells were purchased from Shanghai Weidi; plasmid extraction kits, gel recovery kits, and genome extraction kits were all purchased from Quanshijin Biotechnology Co., Ltd.; and RNA extraction kits were purchased from Tiangen Biochemical Technology Co., Ltd.

[0020] The nomenclature of TrWRKY10a540: Tr stands for white clover ( Trifolium repens L.), WRKY indicates that this gene belongs to the WRKY transcription factor family. The gene is located on chromosome 10 (Chr10) at locus chr10.jg540. TrWRKY10a540 belongs to Group IIa of the WRKY transcription factor family. Its gene structure contains three introns and encodes a 288-amino acid protein.

[0021] The nucleotide sequence of TrWRKY10a540 is shown in SEQ ID NO.1; the amino acid sequence of TrWRKY10a540 is shown in SEQ ID NO.2.

[0022] Example 1. Cloning and sequencing of the TrWRKY10a540 gene.

[0023] 1. Total RNA Extraction and cDNA Synthesis.

[0024] First, total RNA was extracted from fresh white clover (Trifolium repens L.) leaves using an RNA extraction kit according to standard protocols. The extracted RNA was assayed using a NanoDrop 2000 spectrophotometer to ensure purity met the requirements for subsequent experiments. Subsequently, the total RNA was reverse transcribed using the PrimeScript™ RT Kit to synthesize cDNA. This process ensured the absence of genomic DNA contamination.

[0025] 2. PCR amplification of the TrWRKY10a540 gene.

[0026] The cDNA was diluted 5-fold and used as a template. Primer5 software was used to design specific primers TrWRKY10a540-F and TrWRKY10a540-R for the TrWRKY10a540 gene. The open reading frame (ORF) region of the TrWRKY10a540 gene in white clover was amplified by PCR using primers TrWRKY10a540-F and TrWRKY10a540-R. The sequences of primers TrWRKY10a540-F and TrWRKY10a540-R are as follows: TrWRKY10a540-F: AATCAACCAACCCTATAT; TrWRKY10a540-R: ATGAATATGATTGTGTTAT.

[0027] The PCR reaction system (50 μL) was as follows: 1 μL cDNA template, 1 μL each of primers TrWRKY10a540-F and TrWRKY10a540-R, 25 μL PrimeSTAR Max Premix (2X), and 22 μL dd H2O. All loading steps were performed on ice.

[0028] The PCR amplification reaction program was as follows: pre-denaturation at 94°C for 5 min, denaturation at 98°C for 10 s, annealing at 52°C for 15 s, extension at 72°C for 15 s, 35 cycles, and a final extension at 72°C for 10 min.

[0029] The reaction system and procedure ensured efficient amplification of the TrWRKY10a540 gene, and the obtained PCR amplification products were detected by agarose gel electrophoresis to confirm the size and purity of the target band.

[0030] 3. Agarose gel electrophoresis and product purification.

[0031] PCR amplification products were separated by agarose gel electrophoresis. After electrophoresis, the target band was excised and the product was purified using a gel extraction kit. The purified PCR product was ligated into the pMD18-T vector. The ligation reaction system consisted of 2 μL of gel extraction product ligated with 1 μL of pMD18-T. 5 μL of Solution I and 1 μL of ddH2O were added, mixed, and incubated in a PCR instrument at 16°C for 30 minutes to obtain the ligation product, pMD18T-TrWRKY10a540 plasmid. The ligation product was transformed into DH5α competent Escherichia coli for subsequent clone screening.

[0032] 4. Clone screening and sequencing.

[0033] Screen the transformed DH5α E. coli single clones on LB plates containing ampicillin (Amp) antibiotics. Select single colony clones for PCR ( Figure 1 ) and sequencing. Sequencing results were compared using the bioinformatics software DNAMAN to confirm the correctness of the target gene sequence, ultimately obtaining the complete sequence of the TrWRKY10a540 gene. Simultaneously, 300 μL of the verified correct clone solution was mixed with 300 μL of 50% glycerol and stored at -80°C.

[0034] Example 2: Analysis of physicochemical properties and prediction of tertiary structure of the white clover TrWRKY10a540 gene.

[0035] 1. This example analyzes the protein characteristics of the white clover TrWRKY10a540 transcription factor. Using Proparam prediction, it is found that the molecular formula of the protein encoded by TrWRKY10a540 is C 1405 H 2250 N 408 O 453 S 14 The molecular weight is 32554.62 kDa, the theoretical isoelectric point is 6.90; its instability index is 44.24, which indicates that TrWRKY10a540 protein is an unstable acidic protein.

[0036] 2. Subcellular localization prediction by WoLF PSORT showed that the TrWRKY10a540 protein accounted for 61.0%, 34.1% and 4.9% of the nucleus, cell membrane and cytoskeleton, respectively.

[0037] 3. AlphaFold 2 was used to predict the spatial structure of the trimer assembly structure of the protein encoded by the TrWRKY10a540 gene, with pTM=0.62 and ipTM=0.61. The results were visualized using ChimeraX (version 1.9). Figure 2 shown.

[0038] Example 3: Construction of an expression vector for the TrWRKY10a540 gene in plants.

[0039] 1. Using the pMD18T-TrWRKY10a540 plasmid obtained in Example 1 as a template, perform double digestion with BamHI and PstI. Simultaneously, use BamHI and PstI to double digest the pCAMBIA1300 plasmid. The double digestion system is as follows: 3 μL of pMD18T-TrWRKY10a540 or pCAMBIA1300 plasmid, 0.5 μL each of HindIII and BamHI, 1 μL of 1× K buffer, and 5 μL of ddH2O. Incubate in a PCR instrument at 37°C for 30 min.

[0040] 2. After agarose gel electrophoresis, the target gene fragment and vector plasmid were digested with the aforementioned enzymes and the target fragment was recovered. The target gene fragment was ligated to the vector using T4 ligase using the following ligation system: 2 μL pCAMBIA1300, 6 μL TrWRKY10a540 gene fragment, 1 μL T4 ligase, and 1 μL 10× buffer. Ligation was carried out at 16°C for 4-5 h.

[0041] 3. Transform the ligation product obtained above into competent E. coli DH5α cells using the heat shock method. After culturing overnight in LB solid medium containing kanamycin (Kan), identify the cells by colony PCR. Pick a single colony that has been identified and transfer it to liquid LB medium containing Kan. Incubate the culture overnight on a shaker at 37°C and 200 rpm for 12 hours. Extract the plasmid, send it for sequencing, and designate the correct plasmid as pCAMBIA1300-TrWRKY10a540.

[0042] Example 4: Construction and identification of overexpression lines of the TrWRKY10a540 gene infected Arabidopsis thaliana.

[0043] 1. The pCAMBIA1300-TrWRKY10a540 plasmid obtained in Example 3 was transformed into Agrobacterium GV3101 by freeze-thaw method.

[0044] Add 1 μg of recombinant plasmid to 100 μL of GV3101 competent Agrobacterium tuber culture medium. Gently mix and incubate on ice for 10 minutes, quickly freeze in liquid nitrogen for 5 minutes, and then in a 37°C water bath for 5 minutes. After a 5-minute ice bath, add 800 μL of antibiotic-free YEB liquid medium and incubate in a centrifuge tube at 28°C and 200 rpm for 2-3 hours. Then, a 500 μL aliquot of the bacterial suspension was spread onto YEB solid medium supplemented with the corresponding antibiotics (50 mg / L Rif and 50 mg / L Kan). Incubate in an inverted position at 28°C for 48-72 hours. Single colonies were picked and transferred to liquid YEB medium supplemented with 50 mg / L Rif and 50 mg / L Kan, shaken at 28°C and 200 rpm. After the suspension became turbid, PCR analysis was performed. Positive strains were added to glycerol stock and stored at -80°C.

[0045] 2. Agrobacterium-mediated genetic transformation of Arabidopsis thaliana.

[0046] Agrobacterium GV3101 glycerol culture containing the pCAMBIA1300-TrWRKY10a540 overexpression vector stored at ultra-low temperature was activated twice in YEB liquid medium and cultured in a shaker at 28°C and 200 rpm. When the OD value of the bacterial solution reached 0.6-0.8, it was centrifuged at 5000 rpm at room temperature and the supernatant was discarded. The above bacterial solution was precipitated and resuspended in 5% sucrose solution containing 200 μL / L silwet-77 (OD = 0.4-0.5).

[0047] 3. Infection of Arabidopsis inflorescence.

[0048] The above-obtained resuspension was used to infect the Arabidopsis inflorescences. Arabidopsis plants that were about 30 days old, with pods on the main inflorescences and 2-10 cm long side inflorescences and strong growth were selected. The flowering inflorescences and pods of the Arabidopsis were cut off, and the unflowered but white inflorescences were immersed in the above-obtained resuspension for about 30 seconds. After watering, the plants were cultured in the dark for 24 hours and then transferred to normal light culture conditions. The plants after the above treatment were restored to normal light and water and fertilizer management, and continued to be cultured until the seeds matured, and the T0 generation seeds were collected.

[0049] 4. Positive screening of transgenic seedlings.

[0050] The T0 generation seeds obtained above were positively screened. After surface disinfection, the T0 generation seeds were evenly sown on a 1 / 2 solid MS selective medium containing Kan resistance and cultured at 22°C and 16 hours of light for about 7 days. Plants with good growth and developed root systems were selected and transplanted to disinfected nutrient soil for further growth. Full T1 generation seeds were selected and rinsed once with sterile water, then soaked and disinfected with 75% alcohol for 30 seconds, and then thoroughly rinsed with sterile water. They were then disinfected with 1% sodium hypochlorite for 5 minutes and rinsed 5-6 times with sterile water. After disinfection, they were evenly placed on a 1 / 2 solid MS medium containing Kan resistance and vernalized at 4°C for 2 days before cultured under normal conditions (culture conditions were 24 / 22°C (day / night), 60% humidity, 16 h light / 8 h dark). After two weeks of culture, Arabidopsis thaliana with good growth and normal growth were selected and transplanted into plastic pots filled with nutrient soil. PCR verification was performed using Arabidopsis leaf DNA as a template. The results were as follows: Figure 3 As shown, the products consistent with the target bands were sent to BGI for sequencing and alignment; three strains with higher expression levels were selected from the primary positive strains, and self-pollination screening was continued to the T3 generation (named S2, S4 and S7), and stable overexpression transgenic Arabidopsis strain seeds were obtained for subsequent functional analysis and stress resistance experiments.

[0051] Example 5: Application of the TrWRKY10a540 gene in improving plant cold resistance.

[0052] 1. Planting of transgenic Arabidopsis thaliana.

[0053] After disinfection and cleaning, wild-type and T3 transgenic Arabidopsis seeds were planted in 1 / 2 solid MS medium, vernalized for 3 days in the dark at 4°C, and grown in a light incubator for two weeks. Arabidopsis seedlings with consistent growth were selected and planted in small square pots containing nutrient soil with a nutrient soil and vermiculite ratio of 1:1 (culture conditions were 24 / 22°C (day / night), 60% humidity, 16 h light / 8 h dark), and continued to be cultured for 2 weeks. T3 transgenic Arabidopsis and wild-type Arabidopsis were cultured under 4°C low temperature stress and normal culture conditions, and their growth conditions were observed. The results are as follows Figure 4 , the low temperature tolerance of S2, S4 and S7 was significantly improved compared with the wild type.

[0054] The plants were divided into two groups: the control group cultured under normal culture conditions and the experimental group cultured under low temperature stress for 24 h (16 h light / 8 h dark). The samples were collected from each group, quickly frozen in liquid nitrogen, and stored in a -80°C freezer. The relative expression levels of TrWRKY10a540 in transgenic Arabidopsis were analyzed. The results are shown in Figure 2. Figure 5 , gene TrWRKY10a540 was detected in S2, S4, and S7.

[0055] The effects of overexpression of TrWRKY10a540 on the physiological and biochemical parameters of Arabidopsis thaliana under low temperature were examined. The following six parameters were included: chlorophyll (CHL) content, malondialdehyde (MDA) content, proline (Pro) content, catalase (CAT) content, peroxidase (POD) content, and superoxide dismutase (SOD) content. The results are shown in Figure 6 .

[0056] The results showed that under normal temperature conditions, there was no significant difference in the physiological indicators between transgenic and wild-type plants. However, after low temperature stress treatment, the transgenic lines showed significant advantages in multiple indicators. First, the chlorophyll content of transgenic plants was significantly higher than that of wild-type plants ( Figure 6 A), indicating that it can effectively slow down the degradation of chlorophyll under low temperature conditions, which helps to maintain photosynthesis efficiency. Secondly, the MDA content is significantly lower than that of the wild type ( Figure 6 B), indicating that the degree of membrane lipid peroxidation was mild and the cell membrane integrity was less damaged. In addition, the accumulation of proline in the transgenic plants was significantly increased ( Figure 6 C), suggesting that it has stronger adaptability in osmotic adjustment and low temperature stress relief. In terms of antioxidant enzyme activity, the CAT, POD and SOD activities of the transgenic plants were significantly higher than those of the wild type ( Figure 6 D- Figure 6 The results indicate that the TrWRKY10a540 gene is more effective in scavenging reactive oxygen species and alleviating oxidative stress. This series of improvements in physiological indicators comprehensively demonstrates the key role of the TrWRKY10a540 gene in enhancing plant tolerance to low temperature stress.

Claims

1. A gene for improving plant cold resistance, characterized in that: The gene is the white clover TrWRKY10a540 gene.

2. The gene according to claim 1, characterized in that The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

3. The gene according to claim 1, characterized in that The amino acid sequence encoded by the gene is shown in SEQ ID NO.

2.

4. Use of the TrWRKY10a540 gene according to claim 1 in plant resistance to cold stress.

5. An expression vector for improving plant cold resistance, characterized in that The expression vector is the pCAMBIA1300-TrWRKY10a540 expression vector comprising the TrWRKY10a540 gene according to claim 1.

6. Use of the pCAMBIA1300-TrWRKY10a540 expression vector according to claim 5 in plant resistance to cold stress.

7. A breeding method for regulating plant resistance to cold stress, characterized in that: The breeding method steps are as follows: S1: The coding sequence of TrWRKY10a540 from white clover was cloned using primers to obtain the gene clone sequence; S2: Connect the gene clone sequence to the vector pCAMBIA1300 to obtain the expression vector; S3: Introduce the expression vector obtained in S2 into Agrobacterium to obtain recombinant Agrobacterium; S4: Infect plants with the recombinant Agrobacterium obtained in S3 to obtain transgenic plants.

8. The breeding method according to claim 7, characterized in that The Agrobacterium described in S3 is GV3101.

9. The breeding method according to claim 7, characterized in that The plant described in S4 is Arabidopsis thaliana.

10. The breeding method according to claim 7, characterized in that The infection described in S4 is the inflorescence infection method.

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