Application of Trifolium repens TrWRKY7c1 gene in improving plant cold resistance

Abstract

The application of Trifolium repens TrWRKY7c1 gene in improving plant cold resistance belongs to the technical field of plant genetic engineering and crop genetic breeding. In order to solve the technical blank of the existing technology about the research on low-temperature stress related genes of Trifolium repens, the application of Trifolium repens TrWRKY7c1 gene in improving plant cold resistance is provided, the nucleotide sequence of the gene is shown as SEO ID NO. 1, and the amino acid sequence encoded by the gene is shown as SEO ID NO. 2; a recombinant cloning vector, a plant expression vector or a recombinant glycerol preservation strain containing the gene are also provided, and a transgenic plant is obtained through an agrobacterium-mediated genetic transformation method. The TrWRKY7c1 gene provided in the application can be applied to improve plant cold resistance, cultivate cold-resistant plants, and provide a new gene resource for plant cold resistance breeding, and has important significance for the improvement of cold resistance and the research on the regulation mechanism of plant growth and development.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and crop genetic breeding technology, specifically involving the application of the white clover TrWRKY7c1 gene in improving plant cold resistance. Background Technology

[0002] White clover (Trifolium repens L.), a perennial herbaceous plant belonging to the genus Trifolium in the legume family, is one of the most widely cultivated forage crops in the world, characterized by its rapid growth, strong nitrogen-fixing ability, and high adaptability. However, white clover is highly susceptible to abiotic stresses during its growth, especially during the harsh winters in high-latitude regions, which often leads to abnormal mortality, thus severely restricting its production and promotion.

[0003] Plant cold resistance is regulated by multiple genes, and the WRKY transcription factor family plays a key role in plant responses to abiotic stress. Previous studies have found that several WRKY family members can regulate the physiological metabolism and antioxidant system of plants under low-temperature stress. However, there are few reports on low-temperature stress-related genes in white clover in current research. Summary of the Invention

[0004] This invention addresses the technological gap in existing research on genes related to low-temperature stress in white clover by providing the application of the TrWRKY7c1 gene in improving plant cold resistance.

[0005] One of the objectives of this invention is to provide the application of the white clover TrWRKY7c1 gene in improving the cold resistance of plants, wherein the nucleotide sequence of the white clover TrWRKY7c1 gene is shown in SEO ID NO.1.

[0006] In a preferred embodiment of the present invention, the amino acid sequence encoded by the white clover TrWRKY7c1 gene is shown in SEO ID NO.2.

[0007] A second objective of this invention is to provide a recombinant cloning vector containing the white clover TrWRKY7c1 gene with the nucleotide sequence shown in SEO ID NO.1, and the vector for the recombinant cloning vector is pMD. TM 18-TVector Cloning Kit.

[0008] The third objective of this invention is to provide a recombinant plant expression vector containing the white clover TrWRKY7c1 gene with the nucleotide sequence shown in SEO ID NO.1, and the vector of the plant expression vector is pCAMBIA1300.

[0009] The fourth objective of this invention is to provide a recombinant glycerol preservation strain, which is obtained by ligating the host bacterium *Escherichia coli* DH5α with the aforementioned recombinant cloning vector or the aforementioned recombinant plant expression vector.

[0010] In a preferred embodiment of the present invention, the recombinant glycerol preservation strain is preserved in a culture medium containing 50% glycerol.

[0011] The fifth objective of this invention is to provide the application of the above-mentioned recombinant cloning vector, recombinant plant expression vector, or recombinant glycerol-preserved strain in improving plant cold resistance.

[0012] The sixth objective of this invention is to provide a method for cultivating cold-resistant plants, the cultivation method comprising the following steps:

[0013] The above-mentioned recombinant cloning vectors, recombinant plant expression vectors, or recombinant glycerol-preserved strains containing the white clover TrWRKY7c1 gene were transformed into Agrobacterium, and transgenic plants were obtained by inflorescence infection. Plants with significantly enhanced cold resistance under low temperature conditions were obtained through screening.

[0014] In a preferred embodiment of the present invention, the Agrobacterium is GV3101.

[0015] In a preferred embodiment of the present invention, the plant is Arabidopsis thaliana.

[0016] The beneficial effects of the present invention are as follows: The present invention provides the application of the white clover TrWRKY7c1 gene in improving the cold resistance of plants. The nucleotide sequence of the white clover TrWRKY7c1 gene is shown in SEO ID NO.1, and the amino acid sequence it encodes is shown in SEO ID NO.2. The present invention also provides the application of recombinant cloning vectors, plant expression vectors or recombinant glycerol-preserved strains containing this gene in improving the cold resistance of plants.

[0017] Experiments have demonstrated that, after low-temperature treatment, transgenic Arabidopsis thaliana overexpressing the TrWRKY7c1 gene showed better growth and significantly superior physiological and biochemical indicators compared to wild-type Arabidopsis thaliana, indicating that the transgenic Arabidopsis thaliana exhibits greater cold tolerance than wild-type Arabidopsis thaliana. Therefore, the TrWRKY7c1 gene provided by this invention can be used to improve plant cold resistance and cultivate cold-resistant plants, providing a new gene resource for cold-resistance breeding and holding significant importance for research on cold resistance improvement and the regulatory mechanisms of plant growth and development. Attached Figure Description

[0018] Figure 1 Electrophoresis image of the CDS sequence amplification of the TrWRKY7c1 gene;

[0019] Figure 2 Three-dimensional structure diagram of the protein encoded by the TrWRKY7c1 gene;

[0020] Figure 3 This is a graph showing the PCR detection results of T3 generation transgenic plants;

[0021] Figure 4 Phenotypic diagram of transgenic Arabidopsis thaliana under low temperature stress;

[0022] Figure 5 This is a graph showing the relative expression level of the TrWRKY7c1 gene in transgenic Arabidopsis thaliana.

[0023] Figure 6 The graphs show the physiological indicators of transgenic Arabidopsis thaliana under low temperature stress; A is the chlorophyll content detection graph, B is the malondialdehyde content detection graph, C is the proline content detection graph, D is the catalase activity detection graph, E is the peroxidase activity detection graph, and F is the superoxide dismutase activity detection graph. Detailed Implementation

[0024] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0026] The primers used in the following examples were all synthesized by Shanghai Sangon Biotech Co., Ltd.; sequencing was performed by Shanghai Sangon Biotech Co., Ltd.; the restriction endonucleases, ligases, and pMD used in the experiments were all synthesized by Shanghai Sangon Biotech Co., Ltd. TM The 18-T Vector Cloning Kit, DNA Marker, PrimeSTAR Max Premix (2X), and reverse transcription kit were all purchased from Takara Bio Inc.; DH5α competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd.; the ClonExpress II One Step Cloning Kit was purchased from Novizan Biotech Co., Ltd.; and the plasmid extraction kit, gel extraction kit, and genome extraction kit were all purchased from TransGen Biotech Co., Ltd.

[0027] The naming of TrWRKY7c1: Tr represents white clover (Trifolium repens L.), WRKY represents that the gene belongs to the WRKY transcription factor family, 7 represents that it is located on chromosome 7, c represents that the gene belongs to the WRKY IIc subfamily, and 1 represents that the gene is the first member of this subfamily on chromosome 7; the nucleotide sequence of the TrWRKY7c1 gene is shown in SEO ID NO.1, the ORF region of the TrWRKY7c1 gene is 877 bp; the amino acid sequence encoded by the TrWRKY7c1 gene is shown in SEO ID NO.2, the TrWRKY7c1 gene encodes 292 amino acids, and the protein molecular weight is 32394.14 kDa.

[0028] Example 1: Cloning of the TrWRKY7c1 gene

[0029] Total RNA was extracted from fresh white clover tissue using a plant RNA extraction kit. The extracted RNA was then used as a template to perform RNA extraction using PrimeScript. TM cDNA was synthesized by reverse transcription using an RT reagent kit with a gDNA Eraser. The obtained cDNA was diluted 5 times and used as a template. Gene-specific primers for white clover TrWRKY7c1 were designed using Primer5 software. PCR amplification of the open reading frame (ORF) region of the white clover TrWRKY7c1 gene was performed using primers TrWRKY7c1-F1 (nucleotide sequence shown in SEO ID NO.3) and TrWRKY7c1-R1 (nucleotide sequence shown in SEO ID NO.4).

[0030] The PCR amplification reaction system consisted of: 1 μL cDNA template, 1 μL each of upstream and downstream primers, 25 μL PrimeSTAR MaxPremix (2X), and 22 μL dd H2O. All sample loading steps were performed on ice. The PCR amplification reaction program was: 94℃ for 5 min; 98℃ for 10 s, 52℃ for 15 s, 72℃ for 15 s, for 35 cycles; and finally 72℃ for 10 min.

[0031] The PCR amplification products were detected by 1% agarose gel electrophoresis, and the bands were observed in a gel imaging system. The TrWRKY7c1 gene CDS sequence amplification electrophoresis results are as follows: Figure 1 As shown, a band of the same length as the target gene fragment was excised, and the DNA fragment was purified using a gel extraction kit. 2 μL of the recovered product was ligated with 1 μL of pMD18-T, and 5 μL of Solution I and 1 μL of dd H2O were added. After mixing, the mixture was placed in a PCR instrument and reacted at 16℃ for 30 min to obtain the pMD18T-TrWRKY7c1 plasmid.

[0032] The ligation product was transformed into DH5α competent cells via heat shock, and the specific steps are as follows:

[0033] Thaw competent cells in an ice bath, add 10 μL of ligation reaction solution, mix gently, and incubate on ice for 30 min; heat shock at 42℃ for 45 s, then immediately return to ice bath for 2 min; add 700 μL of LB liquid medium preheated at 37℃, and place the mixture on a shaker at 37℃ and 200 rpm for 1-1.5 h; centrifuge at 5000 rpm for 1 min, discard most of the supernatant, and spread evenly on LB plates containing Amp; incubate upside down at 37℃ for 12-16 h, select suitable single colonies, and perform colony PCR identification using cloning primers TrWRKY7c1-F1 (nucleotide sequence shown in SEO ID NO. 3) and TrWRKY7c1-R1 (nucleotide sequence shown in SEO ID NO. 4);

[0034] The colony PCR system (20 μL) is as follows: single colony template, 1 μL 10×PCR Buffer, 0.8 μL dNTP, 0.4 μL each of TrWRKY7c1-F and TrWRKY7c1-R primers, 0.1 rTaq, and 6.3 μL ddH2O; the colony PCR reaction conditions are: 94℃ for 5 min pre-denaturation; 94℃ for 30 s, 52℃ for 1 min, 72℃ for 1 min, for 35 cycles; 72℃ for 10 min extension.

[0035] Select the successfully identified single colonies and place them in LB liquid medium containing ampicillin (Amp). Incubate overnight at 37°C and 200 rpm with shaking for 12-16 hours. Extract positive plasmids according to the instructions of the plasmid extraction kit. The plasmid extraction process was strictly carried out in accordance with the kit instructions. Send 10 μL of plasmid to the biotechnology company for sequencing. Compare the sequencing results with DNAMAN software to verify the sequence correctness. Take 300 μL of the verified correct clone and mix it with 300 μL of 50% glycerol. Store at -80°C.

[0036] Example 2: Physicochemical property analysis and tertiary structure prediction of the TrWRKY7c1 gene in white clover

[0037] This embodiment analyzes the protein characteristics of the white clover TrWRKY7c1 transcription factor. Using Proparam prediction, it was determined that the protein encoded by TrWRKY7c1 has the molecular formula C. 1402 H 2202 N 398 O 457 S 14With a molecular weight of 32394.14 kDa and a theoretical isoelectric point of 6.26, and an instability index of 54.43, it is inferred that the TrWRKY7c1 protein is an unstable acidic protein.

[0038] Subcellular localization prediction using WoLF PSORT showed that the TrWRKY7c1 protein was located in the cell nucleus.

[0039] The spatial structure of the dimer assembly of the protein encoded by the TrWRKY7c1 gene was predicted using AlphaFold 2, with pTM = 0.63 and ipTM = 0.60. The results were visualized using ChimeraX (version 1.9) software. Figure 2 As shown.

[0040] Example 3: Construction of a plant expression vector for the TrWRKY7c1 gene

[0041] Using the pMD18T-TrWRKY7c1 plasmid obtained in Example 1 as a template, double digestion was performed using BamHI and HindIII enzymes. Simultaneously, the pCAMBIA1300 plasmid was also double digested using BamHI and HindIII enzymes. The double digestion system was as follows: 3 μL of pMD18T-TrWRKY7c1 plasmid or pCAMBIA1300 plasmid, 0.5 μL each of HindIII and BamHI, 1 μL of 1×K buffer, and 5 μL of dd H2O. The mixture was incubated at 37°C for 30 min in a PCR instrument.

[0042] The target gene fragment and vector plasmid digested with the above enzymes were subjected to agarose gel electrophoresis, and the target fragment was recovered from the gel. The target gene fragment was ligated into the vector by T4 ligase. The ligation system was as follows: 2 μL pCAMBIA1300, 6 μL L rWRKY7c1 gene fragment, 1 μL T4 ligase, and 1 μL 10× buffer; ligation was carried out at 16℃ for 4-5 h.

[0043] The ligation product obtained above was transformed into Escherichia coli DH5α competent cells using the heat shock method. After being cultured overnight in LB solid medium containing kanamycin (Kan), colony PCR identification was performed. Single colonies that were successfully identified were picked and cultured overnight for 12 hours in LB liquid medium containing Kan at 37°C and 200 rpm on a shaker. The plasmid was extracted and sent for sequencing. The correct plasmid was named pCAMBIA1300-TrWRKY7c1.

[0044] Example 4: Construction and identification of Arabidopsis thaliana overexpression lines infected with the TrWRKY7c1 gene

[0045] The pCAMBIA1300-TrWRKY7c1 plasmid obtained in Example 3 was transformed into Agrobacterium GV3101 using a freeze-thaw method. The specific steps are as follows:

[0046] Add 1 μg of recombinant plasmid to 100 μL of GV3101 Agrobacterium competent cells, mix gently, and then sequentially incubate on ice for 10 min, flash freeze in liquid nitrogen for 5 min, incubate in a 37°C water bath for 5 min, and incubate on ice for 5 min. Add 800 μL of antibiotic-free YEB liquid medium and incubate in centrifuge tubes at 28°C and 200 rpm for 2-3 h with shaking. Spread 500 μL of the bacterial culture onto YEB solid medium containing the corresponding antibiotics (Rif 50 mg / L, Kan 50 mg / L). Incubate at 28°C with the medium inverted for 48-72 h. Pick single colonies and incubate in liquid YEB medium (containing antibiotics Rif 50 mg / L, Kan 50 mg / L) with shaking at 28°C and 200 rpm. After the bacterial culture becomes turbid, perform bacterial PCR detection. Add the positive bacterial cultures that pass the test to glycerol and store at -80°C.

[0047] The specific steps of Agrobacterium-mediated genetic transformation in Arabidopsis thaliana are as follows:

[0048] Agrobacterium GV3101 glycerol bacteria containing the pCAMBIA1300-TrWRKY7c1 overexpression vector, preserved under ultra-low temperature conditions, were activated twice in YEB liquid medium and cultured with shaking at 28℃ and 200 rpm. When the OD value of the bacterial solution reached 0.6-0.8, it was centrifuged at 5000 r / min at room temperature, and the supernatant was discarded. The above bacterial solution was precipitated and resuspended in a 5% sucrose solution containing 200 μL / Lsilwet-77 (OD = 0.4-0.5).

[0049] The Arabidopsis inflorescences were treated with the resuspended solution obtained above. Robust Arabidopsis plants that had grown for about 30 days, with pods already formed in the main inflorescence and lateral inflorescences 2-10 cm in length were selected. The flowering inflorescences and pods were removed, and the unflowered but budding inflorescences were immersed sequentially in the resuspended solution obtained above for about 30 seconds. After watering, the plants were cultured in darkness for 24 hours, then transferred to normal light conditions. The treated plants were then restored to normal light and water / fertilizer management and cultured until the seeds matured. T0 generation seeds were then harvested.

[0050] Positive screening was performed on the transgenic seedlings obtained above. T0 generation seeds, after surface sterilization, were evenly sown on MS selective medium containing 50 mg / L Rif and 50 mg / L Kan, and cultured at 22℃ under 16 hours of light for approximately 7 days. Plants showing good growth and well-developed root systems were selected and transplanted into sterilized nutrient soil for continued growth. Plump T0 generation seeds were first rinsed once with sterile water, then soaked in 75% alcohol for 30 seconds for sterilization, thoroughly rinsed with sterile water, then sterilized with 1% sodium hypochlorite for 5 minutes, and rinsed 5-6 times with sterile water. After sterilization, they were evenly placed in Kan-resistant 1 / 2 solid MS medium and vernalized at 4℃ for 2 days, followed by normal cultivation conditions (24 / 22℃ (day / night), 60% humidity, 16h light / 8h darkness). After two weeks of cultivation, well-grown and healthy Arabidopsis thaliana were transplanted into plastic pots filled with nutrient soil. PCR verification was performed using Arabidopsis thaliana leaf DNA as a template. The results are as follows: Figure 3 As shown, products with the same target band were sent to BGI for sequencing and comparison; three lines with high expression levels were selected from the first generation positive lines and self-crossed and screened to the T3 generation (S2, S4 and S8) to obtain stable overexpression transgenic Arabidopsis lines for subsequent functional analysis and stress resistance-related experiments.

[0051] Example 5: Application of the TrWRKY7c1 gene in improving plant cold resistance

[0052] In this embodiment, wild-type and homozygous T3 transgenic Arabidopsis seeds obtained in Example 4 were disinfected and cleaned, then planted in 1 / 2 solid MS medium and vernalized for 3 days at 4°C in the dark. After growing in a light incubator for two weeks, Arabidopsis seedlings with uniform growth were selected and planted in small square pots containing nutrient soil and vermiculite in a 1:1 ratio (culture conditions: 24 / 22°C (day / night), 60% humidity, 16h light / 8h dark) for another 2 weeks. Then, T3 transgenic Arabidopsis and wild-type Arabidopsis were placed under 4°C low-temperature stress culture and normal culture conditions, respectively, and their growth was observed.

[0053] The results are as follows Figure 4 As shown, after low-temperature treatment, the transgenic Arabidopsis thaliana (S2, S4, and S8) overexpressing the TrWRKY7c1 gene exhibited better growth, while the wild-type Arabidopsis thaliana showed more wilting growth; the transgenic Arabidopsis thaliana showed greater cold tolerance than the wild-type Arabidopsis thaliana. Therefore, the TrWRKY7c1 gene provided by this invention can be used to improve the cold resistance of plants and cultivate cold-resistant plants.

[0054] In this embodiment, the above-mentioned plants were divided into two groups: a control group under normal culture conditions and an experimental group subjected to 24 hours of low-temperature stress (16 hours of light / 8 hours of darkness). Materials were collected from each group, flash-frozen in liquid nitrogen, and stored at -80°C. The relative expression level of the TrWRKY7c1 gene in transgenic Arabidopsis thaliana was analyzed, as well as the effects of overexpression of the TrWRKY7c1 gene on the physiological and biochemical indicators of Arabidopsis thaliana under low temperature, specifically including: chlorophyll (CHL) content, proline (Pro) content, malondialdehyde (MDA) content, catalase (CAT) activity, peroxidase (POD) activity, and superoxide dismutase (SOD) activity. The specific detection steps for the above indicators were as described in the literature "Zhang Xueqi. Analysis of gene expression patterns in white clover in response to low-temperature stress [D]. Harbin Normal University, 2023."

[0055] like Figure 5 As shown, compared with wild-type Arabidopsis, the relative expression level of the TrWRKY7c1 gene in transgenic Arabidopsis is higher; statistical results of physiological indicators of transgenic Arabidopsis under low temperature stress.

[0056] like Figure 6 As shown, under normal temperature conditions, there were no significant differences in chlorophyll content, malondialdehyde (MDA) content, proline content, and antioxidant enzyme (CAT / POD / SOD) activity between the TrWRKY7c1 transgenic Arabidopsis and the wild type. However, under low-temperature stress, the transgenic lines (S2 / S4 / S8) exhibited significant advantages, specifically: chlorophyll decomposition was slowed, maintaining higher photosynthetic efficiency; MDA content decreased, reducing membrane lipid peroxidation damage; proline accumulation was enhanced, improving osmotic regulation capacity; simultaneously, CAT, POD, and SOD activities were significantly increased, effectively scavenging reactive oxygen species and alleviating oxidative stress. Therefore, the above synergistic effects indicate that transgenic Arabidopsis overexpressing the TrWRKY7c1 gene significantly enhances the low-temperature adaptability and stress resistance of Arabidopsis by regulating chlorophyll metabolism, membrane stability, osmotic regulation, and the antioxidant defense system.

[0057] The contents not described in detail in this specification are well-known to those skilled in the art. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. White clover TrWRKY7c1 The application of genes in improving plant cold resistance is characterized by, The white clover TrWRKY7c1 The nucleotide sequence of the gene is shown in SEO ID NO.1, and the plant is Arabidopsis thaliana.

2. The application of a recombinant cloning vector or recombinant plant expression vector in improving plant cold resistance, characterized in that, The plant in question is Arabidopsis thaliana. The recombinant cloning vector contains white clover with the nucleotide sequence shown in SEO ID NO.

1. TrWRKY7c1 The gene, wherein the recombinant cloning vector is a pMD18-T Vector; The recombinant plant expression vector contains white clover with the nucleotide sequence shown in SEO ID NO.

1. TrWRKY7c1 The gene, the plant expression vector is pCAMBIA1300.

3. A method for cultivating cold-resistant plants, characterized in that, The cultivation method includes the following steps: Will contain white clover TrWRKY7c1 The recombinant plant expression vector of the gene was transformed into Agrobacterium, and the transgenic plant was obtained by inoculation method. The transgenic plant was obtained by screening and the plant with significantly enhanced cold resistance under low temperature conditions was obtained. The plant was Arabidopsis thaliana. The recombinant plant expression vector contains white clover with the nucleotide sequence shown in SEO ID NO.

1. TrWRKY7c1 The gene, the plant expression vector is pCAMBIA1300.

4. The cultivation method according to claim 3, characterized in that, The Agrobacterium species in question is GV3101.

Images