Cynanchum thesioides transcription factor CtWRKY41 and application of cynanchum thesioides transcription factor CtWRKY41 in improving plant salt tolerance and delaying flowering time

Abstract

The invention relates to the technical field of gene engineering, in particular to a cynanchum thesioides transcription factor CtWRKY41 and application of the cynanchum thesioides transcription factor CtWRKY41 in improving plant salt tolerance and delaying flowering time. The cynanchum thesioides transcription factor CtWRKY41 provided by the invention is a transcription factor of a WRKY gene family capable of improving the salt tolerance of plants, the salt tolerance of the plants or the yeast can be remarkably improved by expressing the coding gene CtWRKY41 in the plants or the yeast, and a technical support is provided for deeply analyzing the biological functions of the WRKY gene family and improving the stress resistance of crops. Furthermore, the invention also finds that the cynanchum thesioides transcription factor CtWRKY41 can also reduce the sensitivity of plants to exogenous abscisic acid, delay the flowering time of the plants, delay the bolting time of the plants and increase the quantity of rosette leaves of the plants, thereby providing a new biological resource for crop improvement.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly to the transcription factor CtWRKY41 of Cynanchum thesioides and its application in improving plant salt tolerance and delaying flowering time. Background Art

[0002] Cynanchum thesioides is an erect semi-shrub with important economic value. It has strong adaptability and grows rapidly, and is mainly distributed in arid and saline-alkali areas. This plant not only has medicinal, edible and forage values, but also is widely used in wasteland treatment and ecological restoration due to its extremely strong stress resistance.

[0003] Research shows that some WRKY genes participate in abiotic stress responses through the abscisic acid (ABA) signal transduction pathway. This dual function may reflect the adaptive strategies formed by plants during long-term evolution, that is, giving priority to survival rather than reproduction under adverse environmental conditions. However, the research on the salt tolerance function of Cynanchum thesioides is still very limited, and there is a lack of gene resources for regulating the salt tolerance of Cynanchum thesioides. Summary of the Invention

[0004] To solve the above problems, the present invention provides the transcription factor CtWRKY41 of Cynanchum thesioides and its application in improving plant salt tolerance and delaying flowering time. The present invention provides a transcription factor CtWRKY41 involved in the salt tolerance of Cynanchum thesioides, which provides technical support for deeply analyzing WRKY the biological functions of gene families and improving the stress resistance of crops.

[0005] To achieve the above object, the present invention provides the following technical solutions: The present invention provides a transcription factor CtWRKY41 of Cynanchum thesioides, and the amino acid sequence is as shown in SEQ ID NO.1.

[0006] The present invention provides a coding gene of the transcription factor CtWRKY41 of Cynanchum thesioides according to the above technical solution, and the nucleotide sequence is as shown in SEQ ID NO.2.

[0007] The present invention provides a recombinant vector expressing the transcription factor CtWRKY41 of Cynanchum thesioides according to the above technical solution, including a backbone vector and the CtWRKY41 gene inserted into the backbone vector; the CtWRKY41 amino acid sequence encoded by the gene is as shown in SEQ ID NO.1.

[0008] Preferably, the backbone vector is a plant expression vector or a yeast expression vector.

[0009] Preferably, the plant expression vector is the PCAMBIA1300-35S-eGFP vector; the yeast expression vector includes the pGBKT7 vector or the pYES2 vector.

[0010] The present invention provides an engineered bacterium, comprising Agrobacterium and a recombinant vector transfected into Agrobacterium; the recombinant vector is the recombinant vector described in the above technical solution.

[0011] Preferably, the Agrobacterium is Agrobacterium tumefaciens.

[0012] The present invention provides the application of a biological material for increasing the expression level of the Cynanchum thesioides transcription factor CtWRKY41 and / or the coding gene described in the above technical solution in one or more of the following: 1) Improving the salt tolerance of plants; 2) Delaying the flowering time of plants; 3) Delaying the bolting time of plants; 4) Reducing the sensitivity of plants to exogenous abscisic acid; 5) Increasing the number of rosette leaves of plants; 6) Improving the salt tolerance of yeast.

[0013] Preferably, the plants include Cynanchum thesioides or Arabidopsis thaliana.

[0014] Preferably, the biological material is the recombinant vector described in the above technical solution or the engineered bacterium described in the above technical solution.

[0015] Beneficial effects: The present invention provides a Cynanchum thesioides transcription factor CtWRKY41, and the amino acid sequence is as shown in SEQ ID NO.1. The Cynanchum thesioides transcription factor CtWRKY41 provided by the present invention is a transcription factor of a gene family that can improve the salt tolerance of plants. Expressing its coding gene in plants or yeast WRKY can significantly improve the salt tolerance of plants or yeast, providing technical support for in-depth analysis of the biological functions of the gene family and improving the stress resistance of crops. CtWRKY41 WRKY

[0016] Furthermore, the present invention also discovers that the Cynanchum thesioides transcription factor CtWRKY41 can also reduce the sensitivity of plants to exogenous abscisic acid (ABA), delay the flowering time of plants, delay the bolting time of plants, and increase the number of rosette leaves of plants, providing a new biological resource for crop improvement. Description of the Drawings

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0018] Figure 1 It is the subcellular localization result of the CtWRKY41 protein in the leaves of Nicotiana benthamiana; Figure 2 Results of the yeast transcriptional activation experiment in Example 1; Figure 3 Results of the effects of CtWRKY41 protein on yeast growth and salt tolerance; Figure 4 Results of the PCR amplification electrophoresis in Example 3; Figure 5 Results of qRT-PCR detection CtWRKY41 of the relative expression levels of genes; Figure 6 Results of the germination and root length physical maps of different groups in Example 4; Figure 7 Results of the germination rate and root length statistics of different groups in Example 4; Figure 8 Results of the phenotypic analysis, fresh weight and chlorophyll content of Arabidopsis thaliana under salt stress; Figure 9 Results of the determination of various indexes of oxidative damage and antioxidant enzyme activities of Arabidopsis thaliana under salt stress; Figure 10 Results of the expression of related genes under salt stress; Figure 11 Results of the experiment in Example 7. Detailed implementation mode

[0019] The present invention provides a Cynanchum thesioides transcription factor CtWRKY41, and the amino acid sequence is shown in SEQ ID NO.1, specifically as follows: MNCATSWEYKSLINELTQGMEKAKQLRVHLSSTSSDQEFLLQRILSSYEKALLILKWSGSSTGQSQQPTPPGSACGALESSISVDGSPRSEELNRNFRDQQDQNMNASKKRKTLPTWTEQVKVSSDNGLEGPSDDGYSWRKYGQKDILGAKYPRSYYRCTYRHIQNCWATKQVQRSDEDATVFEITYRGVHTCNLAGTSNSVASTASPEKQEVRHKNNCSSNYQLQQPNQMLMNLRANLRVNTGDSENKNSTSPFSFPSTLTYFEDENQYFPISTHVDENLMGTYSPSFISPATSESNYFSVSVCNMNSLGGTHNLQHSESDLTDLISATNSPIRGLDFSIDPAELDPNFNFTRSGFFT.

[0020] The present invention also provides a coding gene for the transcription factor CtWRKY41 of Cynanchum thesioides described in the above technical solution, and the nucleotide sequence is as shown in SEQ ID NO.2, specifically as follows:

[0021] The Cynanchum thesioides transcription factor CtWRKY41 provided by the present invention is a Cynanchum thesioides-derived transcription factor that is localized in the nucleus and has transcriptional activation activity. Expressing its coding gene in plants or yeast CtWRKY41 , can significantly improve the salt tolerance of plants or yeast, can reduce the sensitivity of plants to exogenous abscisic acid (ABA), and plays an important role in integrating ABA signal transduction and salt stress response. The experimental results show that CtWRKY41 by improving the antioxidant capacity and maintaining the photosynthetic efficiency, the salt tolerance of transgenic Arabidopsis thaliana was significantly enhanced; CtWRKY41 by enhancing the antioxidant enzyme activity and osmotic adjustment ability, the accumulation of ROS under salt stress conditions was effectively reduced, thereby reducing oxidative damage and improving the salt tolerance of plants; compared with the wild type, CtWRKY41 in the overexpression lines, the expression levels of antioxidant enzyme-related genes ( AtCAT1 , AtPOD1 and AtAPX1 ) were significantly higher. In addition, the expression levels of abscisic acid signaling pathway-related genes ( AtNCED3 , AtABI1 and AtABI2 ) were also significantly up-regulated. Moreover, the expression levels of the plasma membrane sodium antiporter gene ( AtSOS1 ) and the dehydration-responsive gene ( AtRD22 ) in the overexpression lines were significantly higher than those of the wild type.

[0022] In addition, the Cynanchum thesioides transcription factor CtWRKY41 provided by the present invention can also delay the flowering time of plants, delay the bolting time of plants, and increase the number of rosette leaves of plants. The experimental results show that CtWRKY41 it can coordinately regulate the expression of flowering-related genes through multiple pathways. By up-regulating FLC and down-regulating FT and SOC1 , CtWRKY41 the gene inhibits the flowering process. During the flowering transition, it regulates the photoperiod and gibberellin pathways by up-regulating CO and GA20OX . This complex regulatory network reflects the CtWRKY41 important role in integrating different flowering signaling pathways, ultimately resulting in a delay in the flowering phenotype. This discovery provides new technical support for the role of WRKY transcription factors in the plant flowering regulatory network.

[0023] The present invention provides a recombinant vector expressing the Cynanchum thesioides transcription factor CtWRKY41 described in the above technical solution, including a backbone vector and the CtWRKY41 gene inserted into the backbone vector; the amino acid sequence encoded by the CtWRKY41 gene is shown in SEQ ID NO.1.

[0024] As an implementation manner, the skeletal vector is a plant expression vector or a yeast expression vector.

[0025] As an implementation manner, the plant expression vector is the PCAMBIA1300-35S-eGFP vector; the yeast expression vectors include the pGBKT7 vector or the pYES2 vector. The recombinant vector provided by the present invention can express the Cynanchum thesioides transcription factor CtWRKY41 in plants or yeasts, improve the salt tolerance of plants or yeasts, delay the flowering time of plants, delay the bolting time of plants, reduce the sensitivity of plants to exogenous abscisic acid, and increase the number of rosette leaves of plants.

[0026] The present invention provides an engineered bacterium, including Agrobacterium and a recombinant vector transferred into Agrobacterium; the recombinant vector is the recombinant vector described in the above technical solution.

[0027] As an implementation manner, the Agrobacterium is Agrobacterium tumefaciens. As an implementation manner, the Agrobacterium tumefaciens can be Agrobacterium GV3103.

[0028] Based on the above advantages, the present invention provides the application of a biological material for increasing the expression level of the Cynanchum thesioides transcription factor CtWRKY41 described in the above technical solution and / or the coding gene described in the above technical solution in one or more of the following: 1) Improving the salt tolerance of plants; 2) Delaying the flowering time of plants; 3) Delaying the bolting time of plants; 4) Reducing the sensitivity of plants to exogenous abscisic acid; 5) Increasing the number of rosette leaves of plants; 6) Improving the salt tolerance of yeasts.

[0029] As an implementation manner, the plant includes Cynanchum thesioides or Arabidopsis thaliana. The Cynanchum thesioides transcription factor CtWRKY41 provided by the present invention is a transcription factor cloned from Cynanchum thesioides. Its coding gene can be overexpressed in Cynanchum thesioides to increase the expression level of the Cynanchum thesioides transcription factor CtWRKY41, thereby improving the salt tolerance of Cynanchum thesioides, delaying the flowering time of Cynanchum thesioides, delaying the bolting time of Cynanchum thesioides, reducing the sensitivity of Cynanchum thesioides to exogenous abscisic acid, and increasing the number of rosette leaves of Cynanchum thesioides.

[0030] As an implementation manner, the biological material is the recombinant vector described in the above technical solution or the engineered bacterium described in the above technical solution.

[0031] To further illustrate the present invention, the Cynanchum thesioides transcription factor CtWRKY41 provided by the present invention and its application in improving the salt tolerance of plants and delaying the flowering time will be described in detail below in conjunction with examples and drawings, but they should not be construed as limiting the protection scope of the present invention.

[0032] Preparation Example The primer sequences used in the embodiments of the present invention are shown in Table 1.

[0033] Table 1 Primer sequences corresponding to different genes Note: The primers with the prefix At in the table are all primers for Arabidopsis genes, and the primers with Ct are all primers for Cynanchum thesioides genes; Atactin-F and Atactin-R are primers for Arabidopsis internal reference genes, and ACT7-F and ACT7-F are primers for Cynanchum thesioides internal reference genes.

[0034] Example 1 CtWRKY41 Subcellular localization and transcriptional activation 1. CtWRKY41 Subcellular localization Construct CtWRKY41 A fusion expression vector with green fluorescent protein (GFP), and the steps are as follows: Using Cynanchum thesioides genomic DNA as a template, perform PCR amplification with the primers CtWRKY41-eGFP-F and CtWRKY41-eGFP-R in Table 1 to obtain a target fragment containing CtWRKY41 gene.

[0035] Use the restriction endonucleases SacI and XbaI to double-digest the PCAMBIA1300-35S-eGFP vector (denoted as 35S:eGFP), and recover the linearized PCAMBIA1300-eGFP vector.

[0036] Mix the target fragment containing CtWRKY41 gene and the linearized PCAMBIA1300-35S-eGFP vector for homologous recombination to obtain a recombinant vector (denoted as 35S:CtWRKY41-eGFP).

[0037] Transfer the recombinant vector into Agrobacterium tumefaciens GV3103 to obtain a recombinant Agrobacterium tumefaciens solution.

[0038] Use the resuspension solution to adjust the OD 600 value of the recombinant Agrobacterium tumefaciens solution to 0.4 to obtain an infection solution; the resuspension solution consists of the following components at the following concentrations: MgCl2·6H2O 2.033 g / L, 2-(N-morpholino)ethanesulfonic acid (MES) 2.132 g / L, and acetosyringone 200 μM.

[0039] Label the numbers on the leaves, and use a 1 mL syringe to apply pressure from the back of the leaves to apply the infection solution and the nuclear localization marker mCherry (concentration of OD 600Inject (OD = 0.8) into tobacco leaves, then spray water on the leaves, cover with a fresh-keeping bag, and place in the dark overnight. Open the fresh-keeping bag on the 2nd day and observe under a laser confocal microscope; at the same time, set the empty vector (35S:eGFP) as the control group. The results are shown in Figure 1 , from left to right are: green fluorescent protein, nuclear localization signal, merged image and bright field image, and the scale bar is 20 μm.

[0040] The results showed that the fluorescence signal of the CtWRKY41-GFP fusion protein only appeared in the nucleus, while the fluorescence signal of the empty vector control appeared in both the cytoplasm and the nucleus, indicating that the CtWRKY41 protein was localized in the nucleus.

[0041] 2. To further verify the transcriptional activation characteristics of CtWRKY41, the present invention conducted a yeast transcriptional activation experiment, and the steps are as follows: Construct the CtWRKY41-pGBKT7 recombinant plasmid. The construction method is similar to that of the recombinant vector 35S:CtWRKY41-eGFP in step 1, except that the primers used for amplifying the target fragment are CtWRKY41-BDF and CtWRKY41-BDR in Table 1, replace the PCAMBIA1300-eGFP vector with the pGBKT7 vector, and the restriction endonucleases are EcoRⅠ and BamHⅠ.

[0042] Transform the CtWRKY41-pGBKT7 recombinant plasmid and the empty vector pGBKT7 (negative control) into yeast Y2Hgold respectively, and culture them on a medium lacking tryptophan (SD-Trp) and a medium lacking tryptophan, histidine and adenine (SD-Trp-Ade-His) after transformation. The results are shown in Figure 2 .

[0043] The results showed that both the CtWRKY41-pGBKT7 recombinant plasmid and the empty vector could grow normally on the SD-Trp medium; however, only the yeast cells containing the CtWRKY41-pGBKT7 recombinant plasmid could grow on the SD-Trp-Ade-His medium, indicating that CtWRKY41 has transcriptional activation activity.

[0044] In summary, CtWRKY41 is a transcriptional activator localized in the nucleus.

[0045] Example 2 Overexpression CtWRKY41 Enhanced the resistance of yeast to NaCl In yeast containing CtWRKY41 the overexpression vector (pYES2-CtWRKY41), the effects of the CtWRKY41 protein on yeast growth and stress resistance were analyzed, and the steps are as follows: 1. Construct the overexpression vector of CtWRKY41. The construction method is similar to that of recombinant vector 35S:CtWRKY41-eGFP in step 1 of Example 1, with the difference that the primers used for amplifying the target fragment are pYES2-CtWRKY41-F and pYES2-CtWRKY41-R in Table 1, the PCAMBIA1300-eGFP vector is replaced by the pYES2 vector, and the restriction enzymes are BamHⅠ and EcoRⅠ.

[0046] 2. Take 100 µl of INVSc1 competent cells melted on ice, and sequentially add 2 - 5 µg of pre-cooled target plasmid (pYES2-CtWRKY41 or pYES2), 10 µl of Carrier DNA, and 500 µl of PEG / LiAc, and pipette several times to mix evenly. After a 30-min water bath at 30°C, perform a 15-min water bath at 42°C.

[0047] 3. Centrifuge at 10,000 rpm for 30 s to discard the supernatant, resuspend with 400 µl of ddH2O, and centrifuge at 30 s to discard the supernatant.

[0048] 4. Resuspend with 50 µl of ddH2O. After screening for positive clone colonies on the SD / -Ura plate, inoculate them into 15 mL of SD / -Ura Broth yeast-deficient liquid medium and culture overnight at 30°C on a shaker.

[0049] 5. Centrifuge at 1,500 g for 5 min at 4°C to remove the supernatant.

[0050] 6. Resuspend the cell pellet with 50 mL of SG / -Ura liquid medium and culture on a shaker at 30°C.

[0051] 7. Adjust the OD 600 to 0.6. After serial dilution by 10-fold, evaluate the growth of yeast on SD / -Ura Broth yeast-deficient solid medium containing different concentrations of NaCl (0 mM (recorded as control), 500 mM, 1 M). The results are shown in Figure 3 , where A, C, and E are the results of solid medium, and B, D, and F are the results of liquid medium.

[0052] The results show that CtWRKY41 overexpression has no significant effect on the growth of yeast ( Figure 3 A in CtWRKY41 ). The overexpressed yeast cells show significant resistance in the medium containing NaCl (500 mM and 1 M), especially in the case of 1000-fold dilution ( Figure 3 C, E in CtWRKY41After the viability assay of the yeast, it was found that under normal and salt - stress - free conditions, the growth trends of the two yeast strains were basically the same ( Figure 3 in B). Under different concentrations of NaCl stress, CtWRKY41 the viability of the over - expressed yeast strain was always higher than that of the control group. Specifically, at the stress levels of 500 mM and 1 M NaCl, after 9 h of stress treatment, CtWRKY41 the viability of the over - expressed yeast strain was significantly higher than that of the empty vector control strain ( Figure 3 in D, F). These results indicate that the CtWRKY41 protein enhances the ability of yeast to resist salt stress.

[0053] Example 3 CtWRKY41 Heterologous transformation of Arabidopsis thaliana with the gene and screening of positive plants Using the method of Example 1, a CtWRKY41 plant expression vector (i.e., 35S:CtWRKY41 - eGFP) was constructed and transformed into Arabidopsis thaliana by the floral dip method mediated by Agrobacterium tumefaciens GV3103; the wild - type Arabidopsis thaliana used in this invention is the Columbia ecotype (Col - 0). T0 generation transgenic plants were screened on 1 / 2 MS medium containing 50 mg / L HYG. T1 and T2 generations were continuously screened by the same method, and finally, homozygous transgenic lines were obtained.

[0054] To verify the transgenic plants, in this example, genomic DNA of homozygous CtWRKY41 transgenic Arabidopsis thaliana and wild - type Arabidopsis thaliana was extracted, and specific primers (CtWRKY41 - eGFP - F and CtWRKY41 - eGFP - R in Table 1) were used for PCR amplification. The results are as CtWRKY41 shown, where +: the amplification product using the Figure 4 plant expression vector as a template; OE1 - 7: CtWRKY41 transgenic lines; WT: wild - type Arabidopsis thaliana. After identification, OE1, OE2, OE4, OE6, and OE7 were identified as positive plants. CtWRKY41

[0055] Meanwhile, total RNA of Arabidopsis thaliana transgenic homozygous lines that were positive in both HYG screening and molecular identification was extracted. Using the primers CtWRKY41 - qPCR - F and CtWRKY41 - qPCR - R in Table 1, the expression level of the CtWRKY41 gene was detected by qRT - PCR. The primers for the internal reference gene were Atactin - F and Atactin - R in Table 1. The results are shown in Figure 5 p , where ** indicates a significant difference compared with WT ( p <0.01). It can be seen that OE1, OE2, OE4, OE6, and OE7 plants were successfully heterologously transformed CtWRKY41Positive plants of the gene provide reliable experimental materials for subsequent functional analysis.

[0056] Example 4 CtWRKY41 Involved in abscisic acid (ABA) response and enhanced salt tolerance of Arabidopsis thaliana To determine CtWRKY41 whether it is involved in the ABA signaling pathway, this example first analyzed the sensitivity of the transgenic lines screened in Example 3 to ABA treatment through a seed germination experiment. CtWRKY41 Transgenic lines to ABA treatment.

[0057] Collect the mature T3-generation seeds and wild-type (WT) Arabidopsis thaliana seeds, air-dry them, and perform vernalization treatment at 4°C for 2 d (in a 4°C incubator). After vernalization treatment, continuously treat with 70% ethanol for 10 min, and then continuously treat with absolute ethanol for 20 min to surface-sterilize the seeds. Sow the sterilized Arabidopsis thaliana seeds on 1 / 2 MS solid medium (10 cm × 10 cm square plate), and add 150 mM NaCl and 0.6 μM ABA (denoted as the ABA group). At the same time, set up a stress group with only 150 mM NaCl added (denoted as the NaCl group), and a control group without NaCl and ABA added. Place the three groups of seeds in a growth chamber at 24°C, with a 16-h light period and an 8-h dark period, and a relative humidity of 70% for germination and root length tests. Each group is subjected to 3 parallel repeated experiments. Record the germination rate every 12 h, and measure the root length with a vernier caliper after 14 d. The results are shown in Figure 6 and Figure 7 , where Figure 6 the left figure in is the result of the germination test, and the right figure is the result of the root length test; Figure 7 in, ** indicates a significant difference compared with WT ( p < 0.01).

[0058] The results show that treatment with 0.6 μM ABA significantly reduced CtWRKY41 the seed germination rates of transgenic lines and wild-type plants, but the decrease in the seed germination rate of wild-type plants was more obvious. Under salt stress conditions, although NaCl treatment inhibited the germination rate of Arabidopsis thaliana seeds and prolonged their germination time, the germination rate of transgenic plant seeds was significantly higher than that of wild-type plants. Under normal growth conditions, CtWRKY41 promoted the development of lateral roots in overexpression (OE) lines. After treatment with 0.6 μM ABA for 7 d, the main root length of transgenic lines was significantly longer than that of wild-type plants. After being treated with 150 mM NaCl stress, although the root growth of both wild-type and overexpression lines was inhibited, the degree of inhibition of overexpression lines was significantly lighter. These results indicate that CtWRKY41 overexpression of not only reduced the sensitivity of Arabidopsis thaliana to exogenous ABA, but also significantly enhanced the resistance of plants to salt stress. This indicates that CtWRKY41It plays an important role in integrating ABA signaling and salt stress response.

[0059] Example 5 CtWRKY41 Enhanced the physiological indicators and antioxidant capacity of transgenic Arabidopsis To fully evaluate CtWRKY41 The role of salt tolerance in Arabidopsis thaliana, this example is planted in nutrient soil in Example 3 screened CtWRKY41 Transgenic lines were subjected to salt stress treatment as follows: Mature T3 seeds and wild-type (WT) Arabidopsis seeds were collected, air-dried, and vernalized at 4°C. Before sowing on 1 / 2MS solid medium, the seeds were surface-sterilized by continuous treatment with 70% ethanol for 10 min and then with anhydrous ethanol for 20 min. The sterilized seeds were sown in 1 / 2MS medium and transferred to a growth chamber at 24°C, with 16 h of light, 8 h of darkness, and a relative humidity of 70%. When there were four true leaves, they were transplanted to nutrient soil: vermiculite = 1:3 (v / v). Salt stress was applied 14 days after transplanting, and the plants were irrigated with 150 mM NaCl solution. A control group without salt stress was set up at the same time. After 14 days of cultivation, the performance was observed, the fresh weight was recorded, and leaf samples were collected for histochemical staining with diaminobenzidine (DAB, brown) and nitro blue tetrazolium chloride (NBT, blue) to detect the accumulation of reactive oxygen species (ROS) and determine the chlorophyll content. Results Figure 8 , where A: growth phenotype of Arabidopsis thaliana, B: chemical staining results, C: leaf fresh weight, D: total chlorophyll content, ** indicates significant difference compared with WT ( p <0.01).

[0060] The results showed that under normal growth conditions, there were no significant differences in growth status and phenotype between the wild type and overexpression lines. However, after salt stress treatment, the growth of both the wild type and overexpression lines was significantly inhibited, manifested as stunted growth and dehydration and yellowing of leaves, but the inhibition of the overexpression line was less severe ( Figure 8 Middle A).

[0061] Under normal conditions, there was no significant difference in leaf staining between the wild type and the overexpression lines. However, under salt stress conditions, the overexpression lines showed less blue and brown precipitates compared to the wild type ( Figure 8 Figure B), which shows that its hydrogen peroxide (H2O2) and superoxide anion (O2 - ) had a lower accumulation. In addition, the fresh weight and chlorophyll content of the overexpression strain were significantly higher than those of the wild-type strain ( Figure 8 (C, D).

[0062] These results indicate thatCtWRKY41 The salt tolerance of transgenic Arabidopsis thaliana was significantly enhanced by improving its antioxidant capacity and maintaining photosynthetic efficiency.

[0063] Salt stress usually leads to excessive accumulation of reactive oxygen species (ROS) in plants, causing serious oxidative damage. In this example, kits from Beijing Solarbio Science & Technology Co., Ltd. were also used to detect the contents of superoxide anion (H2O2), hydrogen peroxide (O2 - ), malondialdehyde (MDA), the activity of catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), and the content of proline (Pro) in Arabidopsis thaliana leaves. The results are shown in Figure 9, where ** indicates significant difference compared with WT ( p < 0.01).

[0064] The results showed that salt stress significantly increased the levels of malondialdehyde, superoxide anion, and hydrogen peroxide in Arabidopsis thaliana leaves. Compared with wild-type plants, the accumulation amounts of malondialdehyde, superoxide anion, and hydrogen peroxide in overexpression lines were significantly lower ( Figure 9 in A - C of Figure). Salt stress significantly increased the activities of superoxide dismutase, catalase, and peroxidase in both wild-type and overexpression lines, and also increased the accumulation amount of proline. Compared with the wild type, the activities of these antioxidant enzymes and the content of proline in overexpression lines were significantly higher ( Figure 9 in D - G of Figure).

[0065] These results indicate that CtWRKY41 by enhancing the activities of antioxidant enzymes and osmotic adjustment ability, the accumulation of ROS under salt stress conditions was effectively reduced, thereby alleviating oxidative damage and improving the salt tolerance of plants.

[0066] Example 6 CtWRKY41 Regulating the expression of stress-responsive genes To clarify CtWRKY41 the molecular mechanism of enhancing the salt tolerance of Arabidopsis thaliana, in this example, RT-PCR was used with the primers in Table 1 to analyze the relative expression levels of stress-responsive genes in wild-type and overexpression lines under salt stress. The results are shown in Figure 10 .

[0067] The results showed that salt stress significantly induced the expression of multiple key genes in overexpression lines. Compared with the wild type, the expression levels of antioxidant enzyme-related genes ( AtCAT1 , AtPOD1 and AtAPX1 ) in overexpression lines were significantly higher. In addition, the expression levels of genes related to the abscisic acid signaling pathway ( AtNCED3 , AtABI1 and AtABI2 ) were also significantly up-regulated. Moreover, the expression level of the plasma membrane sodium antiporter gene (AtSOS1 ), and the expression levels of dehydration-responsive genes ( AtRD22 ) in the overexpression lines were significantly higher than those in the wild type.

[0068] Example 7 CtWRKY41 Delaying the flowering time of transgenic Arabidopsis During the experiment, the present invention observed that CtWRKY41 is involved in regulating the flowering time of Arabidopsis. In this example, a systematic analysis was carried out on CtWRKY41 the effect on the flowering time: Seeds of wild type and the CtWRKY41 overexpression lines screened in Example 3 were vernalized and then cultured under 16 h light / 8 h dark conditions. The flowering time was recorded by observing the emergence of flower buds, and the corresponding number of rosette leaves was counted. The expression levels of several key flowering-related genes in wild type and CtWRKY41 the overexpression lines were analyzed by qRT-PCR. The results are shown in Figure 11 , where, A: Phenotype of Arabidopsis, scale bar is 2 cm, B: Number of rosette leaves at flowering, C: Time of opening of the first flower in different lines, D: Bolting time (i.e., the time when the inflorescence stem reaches a height of 1 cm), E: Expression levels of endogenous flowering time-related genes in transgenic Arabidopsis overexpressing CtWRKY41 ; ** indicates significant difference compared with WT ( p < 0.01).

[0069] The results showed that the bolting time of wild-type plants was 30.5 d, the flowering time was 34.3 d, and the number of rosette leaves was 12. In contrast, CtWRKY41 the bolting time of the overexpression lines was significantly delayed to 34 d, the flowering time was postponed to 37.8 d, and the number of rosette leaves increased to 17.5. These results indicate that CtWRKY41 overexpression significantly delayed the flowering time of Arabidopsis, which means that CtWRKY41 the gene plays an important role in integrating environmental signals and regulating the flowering time.

[0070] Compared with the wild type, CtWRKY41 the expression of the flowering integrator gene FT (FLOWERING LOCUS T) in the overexpression lines was significantly down-regulated, while the expression of the flowering repressor gene FLC (FLOWERING LOCUS C) was significantly up-regulated. In addition, the expression of the key gene CO (CONSTANS) in the photoperiod pathway and the key gene GA20OX (gibberellin 20-oxidase) in gibberellin biosynthesis was significantly up-regulated in the CtWRKY41 overexpression lines, while the flowering integrator gene SOC1The expression of (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1) was significantly down-regulated.

[0071] These results indicate that CtWRKY41 the expression of flowering-related genes can be coordinately regulated through multiple pathways. By up-regulating FLC and down-regulating FT and SOC1 , CtWRKY41 the gene inhibits the flowering process. During the flowering transition, it regulates the photoperiod and gibberellin pathways by up-regulating CO and GA20OX . This complex regulatory network reflects CtWRKY41 the important role of

[0072] in integrating different flowering signaling pathways, ultimately resulting in a delay in the flowering phenotype. This discovery provides new technical support for the role of WRKY transcription factors in the plant flowering regulatory network. Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A transcription factor of Cucurbita oleracea L., CtWRKY41, characterized in that: The amino acid sequence is shown in SEQ ID NO.

1.

2. A gene encoding the transcription factor CtWRKY41 of claim 1, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

2.

3. A recombinant vector expressing the transcription factor CtWRKY41 of claim 1, characterized in that: comprising a backbone vector and a CtWRKY41 gene; CtWRKY41 The amino acid sequence encoded by the gene is shown in SEQ ID NO.

1.

4. The recombinant vector according to claim 3, characterized in that The backbone vector is a plant expression vector or a yeast expression vector.

5. The recombinant vector according to claim 4, characterized in that The plant expression vector is a PCAMBIA1300-35S-eGFP vector; the yeast expression vector includes a pGBKT7 vector or a pYES2 vector.

6. An engineered bacterium, characterized in that: The invention comprises Agrobacterium and a recombinant vector transformed into Agrobacterium; the recombinant vector is the recombinant vector according to any one of claims 3 to 5.

7. The engineered bacteria according to claim 6, characterized in that The Agrobacterium is Agrobacterium tumefaciens.

8. Use of a biomaterial for increasing the expression of the transcription factor CtWRKY41 of claim 1 and / or the coding gene of claim 2 in one or more of the following: 1) Improve the salt tolerance of plants; 2) Delay the flowering time of plants; 3) Delay the bolting time of plants; 4) Reduce the sensitivity of plants to exogenous abscisic acid; 5) Increase the number of rosette leaves of plants; 6) Improve the salt tolerance of yeast.

9. The use according to claim 8, characterized in that: The plant includes Cucurbita oleracea or Arabidopsis thaliana.

10. The use according to claim 8, characterized in that: The biological material is the recombinant vector according to any one of claims 3 to 5 or the engineered bacteria according to claim 6 or 7.

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