The transcription factor CtWRKY41 of Cucurbita oleracea and its application in improving plant salt tolerance and delaying flowering time

Abstract

The present invention relates to the field of genetic engineering technology, in particular to a transcription factor CtWRKY41 of ground cucumber and its application in improving plant salt tolerance and delaying flowering time. WRKY Transcription factors of a gene family that express their encoding genes in plants or yeast CtWRKY41 , can significantly improve the salt tolerance of plants or yeast, providing a basis for in-depth analysis WRKY This study provides technical support for the biological functions of this gene family and for improving crop stress resistance. Furthermore, the present invention also discovered that the ground melon transcription factor CtWRKY41 can reduce the plant's sensitivity to exogenous abscisic acid, delay the plant's flowering time, delay the plant's bolting time, and increase the number of rosette leaves in the plant, providing a new biological resource for crop improvement.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, in particular to a ground cucumber transcription factor CtWRKY41 and an application thereof in improving plant salt tolerance and delaying flowering time. Background Art

[0002] The ground melon is an upright, economically important semi-shrub with strong adaptability and rapid growth, primarily found in arid, saline-alkali regions. The plant not only has medicinal, edible, and feed properties, but is also widely used in wasteland management and ecological restoration due to its exceptional resilience.

[0003] Studies have shown that some WRKY Genes involved in abiotic stress responses through the abscisic acid (ABA) signaling pathway may reflect an adaptive strategy developed by plants over long periods of evolution, prioritizing survival over reproduction under adverse environmental conditions. However, research on the salt tolerance function of ground cucumber remains limited, and there is a lack of genetic resources regulating salt tolerance in ground cucumber. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides a transcription factor CtWRKY41 of ground cucumber and its application in improving plant salt tolerance and delaying flowering time. WRKY It provides technical support for the biological functions of gene families and improving crop stress resistance.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a radix cucurbitae transcription factor CtWRKY41, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] The present invention provides a gene encoding the cucurbitaceae transcription factor CtWRKY41 described in the above technical solution, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0008] The present invention provides a recombinant vector expressing the radix cucurbitae transcription factor CtWRKY41 described in the above technical solution, comprising a backbone vector and a recombinant vector inserted into the backbone vector. CtWRKY41 gene; CtWRKY41 The amino acid sequence encoded by the gene is shown in SEQ ID NO.1.

[0009] Preferably, the backbone vector is a plant expression vector or a yeast expression vector.

[0010] Preferably, the plant expression vector is a PCAMBIA1300-35S-eGFP vector; the yeast expression vector includes a pGBKT7 vector or a pYES2 vector.

[0011] The present invention provides an engineered bacterium, comprising Agrobacterium and a recombinant vector transformed into the Agrobacterium; the recombinant vector is the recombinant vector described in the above technical solution.

[0012] Preferably, the Agrobacterium is Agrobacterium tumefaciens.

[0013] The present invention provides the use of a biomaterial for increasing the expression of the radix cucurbitae transcription factor CtWRKY41 described in the above technical solution and / or the encoding gene described in the above technical solution in one or more of the following:

[0014] 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.

[0015] Preferably, the plant includes Cucurbita oleracea or Arabidopsis thaliana.

[0016] Preferably, the biological material is the recombinant vector described in the above technical solution or the engineered bacteria described in the above technical solution.

[0017] Beneficial effects:

[0018] The present invention provides a transcription factor CtWRKY41 of ground cucumber, the amino acid sequence of which is shown in SEQ ID NO.1. The transcription factor CtWRKY41 of ground cucumber provided by the present invention is a transcription factor that can improve the salt tolerance of plants. WRKY Transcription factors of a gene family that express their encoding genes in plants or yeast CtWRKY41 , can significantly improve the salt tolerance of plants or yeast, providing a basis for in-depth analysis WRKY It provides technical support for the biological functions of gene families and improving crop stress resistance.

[0019] Furthermore, the present invention also found that the transcription factor CtWRKY41 of the ground melon 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0021] Figure 1The results of subcellular localization of CtWRKY41 protein in Nicotiana benthamiana leaves are shown;

[0022] Figure 2 The yeast transcription activation experiment results in Example 1;

[0023] Figure 3 The results show the effects of CtWRKY41 protein on yeast growth and salt tolerance;

[0024] Figure 4 The results of PCR amplification and electrophoresis in Example 3 are as follows;

[0025] Figure 5 For qRT-PCR detection CtWRKY41 Results of relative gene expression;

[0026] Figure 6 The germination results and root lengths of different groups in Example 4 are shown in FIG.

[0027] Figure 7 The statistical results of germination rate and root length of different groups in Example 4 are shown;

[0028] Figure 8 The results are as follows: phenotypic analysis, fresh weight and chlorophyll content of Arabidopsis thaliana under salt stress;

[0029] Figure 9 The results of the determination of various indicators of oxidative damage and antioxidant enzyme activity in Arabidopsis thaliana under salt stress;

[0030] Figure 10 is the expression result of related genes under salt stress;

[0031] Figure 11 These are the experimental results of Example 7. DETAILED DESCRIPTION

[0032] The present invention provides a transcription factor CtWRKY41 of Cucurbita oleracea, the amino acid sequence of which is shown in SEQ ID NO.1, and is as follows:

[0033] MNCATSWEYKSLINELTQGMEKAKQLRVHLSSTSSDQEFLLQRILSSYEKALLILKWSGSSTGQSQQPTPPGSACGALESSISVDGSPRSEELNRNFRDQQDQNMNASKKRKTLPTWTEQVKVSSDNGLEGPSDDGYSWRKYGQKDILGAKYPRSYYRCTYRHIQNCWATKQVQRSDEDA TVFEITYRGVHTCNLAGTSNSVASTASPEKQEVRHKNNCSSNYQLQQPNQMLMNLRANLRVNTGDSENKNSTSPFSFPSTLTYFEDENQYFPISTHVDENLMGTYSPSFISPATSESNYFSVSVCNMNSLGGTHNLQHSESDLTDLISATNSPIRGLDFSIDPAELDPNFNFFTRSGFFT.

[0034] The present invention also provides a gene encoding the radix cucurbitae transcription factor CtWRKY41 described in the above technical solution, the nucleotide sequence of which is shown in SEQ ID NO.2, specifically as follows:

[0035]

[0036] The present invention provides a transcription factor CtWRKY41 of the ground melon, which is located in the cell nucleus and has transcriptional activation activity. The coding gene thereof is expressed 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 The salt tolerance of transgenic Arabidopsis thaliana was significantly enhanced by improving antioxidant capacity and maintaining photosynthetic efficiency; CtWRKY41 By enhancing the activity of antioxidant enzymes and osmotic regulation ability, the accumulation of ROS under salt stress conditions was effectively reduced, thereby alleviating oxidative damage and improving the salt tolerance of plants; compared with the wild type, CtWRKY41 Antioxidant enzyme-related genes in overexpression lines ( AtCAT1 、 AtPOD1 and AtAPX1 ) were significantly more expressed. In addition, the expression of genes related to the abscisic acid signaling pathway ( AtNCED3 、 AtABI1 and AtABI2 ) was also significantly upregulated. Moreover, the expression level of the plasma membrane sodium antiporter gene ( AtSOS1 ) and dehydration-responsive genes ( AtRD22 ) was expressed at a significantly higher level in the overexpression line than in the wild type.

[0037] In addition, the 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. CtWRKY41 The expression of flowering-related genes can be synergistically regulated through multiple pathways. FLC and lower FT and SOC1 , CtWRKY41 The gene represses the flowering process. During the flowering transition, it is upregulated by CO and GA20OX to regulate the photoperiod and gibberellin pathways. This complex regulatory network reflects CtWRKY41 The discovery of WRKY transcription factors plays an important role in integrating different flowering signaling pathways, ultimately leading to a delayed flowering phenotype. This discovery provides new technical support for the role of WRKY transcription factors in the plant flowering regulatory network.

[0038] The present invention provides a recombinant vector expressing the radix cucurbitae transcription factor CtWRKY41 described in the above technical solution, comprising a backbone vector and a recombinant vector inserted into the backbone vector. CtWRKY41 gene; CtWRKY41 The amino acid sequence encoded by the gene is shown in SEQ ID NO.1.

[0039] As an embodiment, the backbone vector is a plant expression vector or a yeast expression vector.

[0040] In one embodiment, the plant expression vector is a pCAMBIA1300-35S-eGFP vector; the yeast expression vector includes a pGBKT7 vector or a pYES2 vector. The recombinant vector provided by the present invention can express the CtWRKY41 transcription factor of the ground cucumber in plants or yeast, thereby improving the salt tolerance of the plants or yeast, delaying the flowering time of the plants, delaying the bolting time of the plants, reducing the sensitivity of the plants to exogenous abscisic acid, and increasing the number of rosette leaves in the plants.

[0041] The present invention provides an engineered bacterium, comprising Agrobacterium and a recombinant vector transformed into the Agrobacterium; the recombinant vector is the recombinant vector described in the above technical solution.

[0042] In one embodiment, the Agrobacterium is Agrobacterium tumefaciens. In one embodiment, the Agrobacterium tumefaciens may be Agrobacterium GV3103.

[0043] Based on the above advantages, the present invention provides the use of a biomaterial for increasing the expression of the CtWRKY41 transcription factor of the above technical solution and / or the encoding gene of the above technical solution in one or more of the following:

[0044] 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.

[0045] In one embodiment, the plant includes cucurbita or Arabidopsis thaliana. The cucurbita transcription factor CtWRKY41 provided by the present invention is a transcription factor cloned from cucurbita, and its encoding gene can be overexpressed in cucurbita to increase the expression level of the cucurbita transcription factor CtWRKY41, thereby improving the salt tolerance of cucurbita, delaying the flowering time of cucurbita, delaying the bolting time of cucurbita, reducing the sensitivity of cucurbita to exogenous abscisic acid, and increasing the number of rosette leaves of cucurbita.

[0046] As an embodiment, the biological material is the recombinant vector described in the above technical solution or the engineered bacteria described in the above technical solution.

[0047] To further illustrate the present invention, the following detailed description of the C. scoparia transcription factor CtWRKY41 provided by the present invention and its application in improving plant salt tolerance and delaying flowering time is given in conjunction with the examples and drawings, but they should not be construed as limiting the scope of protection of the present invention.

[0048] Preparation Example

[0049] The primer sequences used in the examples of the present invention are shown in Table 1.

[0050] Table 1 Primer sequences corresponding to different genes

[0051]

[0052] Note: Primers prefixed with "At" in the table are primers for Arabidopsis genes, and "Ct" are primers for Cucurbita edulis genes; Atactin-F and Atactin-R are Arabidopsis internal reference gene primers, and ACT7-F and ACT7-F are Cucurbita edulis internal reference gene primers.

[0053] Example 1 CtWRKY41 Subcellular localization and transcriptional activation

[0054] 1. CtWRKY41 Subcellular localization

[0055] Build CtWRKY41 The fusion expression vector with green fluorescent protein (GFP) is as follows:

[0056] Using the genomic DNA of Cucurbita scoparia as a template, PCR amplification was performed using the primers CtWRKY41-eGFP-F and CtWRKY41-eGFP-R in Table 1 to obtain the CtWRKY41 Target gene fragment.

[0057] The PCAMBIA1300-35S-eGFP vector (denoted as 35S:eGFP) was double-digested with restriction endonucleases SacI and XbaI, and the linearized PCAMBIA1300-eGFP vector was recovered.

[0058] The said CtWRKY41 The target gene fragment and the linearized PCAMBIA1300-35S-eGFP vector were homologously recombined to obtain a recombinant vector (denoted as 35S:CtWRKY41-eGFP).

[0059] The recombinant vector is transferred into Agrobacterium GV3103 to obtain a recombinant Agrobacterium bacterial liquid.

[0060] The OD value of the recombinant Agrobacterium solution was adjusted by resuspension 600 The value was adjusted to 0.4 to obtain an infection solution; the resuspension solution was composed of the following components: MgCl2·6H2O 2.033 g / L, 2-(N-morpholino)ethanesulfonic acid (MES) 2.132 g / L and acetosyringone 200 μM.

[0061] Mark the leaves with numbers and use a 1 mL syringe to inject the infection solution and the nuclear localization marker mCherry (concentration OD 600 = 0.8) were injected into tobacco leaves, which were then sprayed with water, covered with a fresh-keeping bag, and left in the dark overnight. The fresh-keeping bag was opened on the second day and observed under a laser confocal microscope. An empty vector (35S:eGFP) was also set up as a control group. Figure 1 , from left to right: green fluorescent protein, nuclear localization signal, merged image and bright field image, scale bar is 20 μm.

[0062] The results showed that the fluorescence signal of the CtWRKY41-GFP fusion protein appeared only in the cell nucleus, while the fluorescence signal of the empty vector control appeared in both the cytoplasm and the cell nucleus, indicating that the CtWRKY41 protein was localized in the cell nucleus.

[0063] 2. To further verify the transcriptional activation properties of CtWRKY41, the present invention conducted a yeast transcriptional activation experiment, the steps are as follows:

[0064] The CtWRKY41-pGBKT7 recombinant plasmid was constructed using a method similar to that used for the recombinant vector 35S:CtWRKY41-eGFP in step 1. The difference was that the primers used to amplify the target fragment were CtWRKY41-BDF and CtWRKY41-BDR listed in Table 1, the PCAMBIA1300-eGFP vector was replaced with the pGBKT7 vector, and the restriction endonucleases were EcoRI and BamHI.

[0065] The CtWRKY41-pGBKT7 recombinant plasmid and the empty vector pGBKT7 (negative control) were transformed into yeast Y2Hgold, and then cultured in a medium lacking tryptophan (SD-Trp) and a medium lacking tryptophan, histidine, and adenine (SD-Trp-Ade-His). Figure 2 .

[0066] The results showed that both the CtWRKY41-pGBKT7 recombinant plasmid and the empty vector could grow normally in SD-Trp medium; however, only yeast cells containing the CtWRKY41-pGBKT7 recombinant plasmid could grow in SD-Trp-Ade-His medium, indicating that CtWRKY41 has transcriptional activation activity.

[0067] In summary, CtWRKY41 is a transcriptional activator localized in the cell nucleus.

[0068] Example 2 Overexpression CtWRKY41 Enhanced yeast resistance to NaCl

[0069] In containing CtWRKY41 The effects of CtWRKY41 protein on yeast growth and stress resistance were analyzed in yeast overexpressing the pYES2-CtWRKY41 vector as follows:

[0070] 1. Construct a CtWRKY41 overexpression vector. The construction method is similar to the recombinant vector 35S:CtWRKY41-eGFP in step 1 of Example 1, except 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 endonucleases are BamHI and EcoRI.

[0071] 2. Take 100µl of INVSc1 competent cells thawed on ice, add 2-5µg of pre-chilled target plasmid (pYES2-CtWRKY41 or pYES2), 10µl of carrier DNA, and 500µl of PEG / LiAc, mix well by pipetting several times, and incubate in a 30°C water bath for 30 minutes, then in a 42°C water bath for 15 minutes.

[0072] 3. Centrifuge at 10,000 rpm for 30 seconds and discard the supernatant. Resuspend in 400 µl of ddH2O and centrifuge for 30 seconds and discard the supernatant.

[0073] 4. Resuspend the plate in 50µl of ddH2O, plate the plasmid onto SD / -Ura plates to screen for positive clones, then inoculate into 15mL of SD / -Ura Broth yeast-deficient liquid medium and culture overnight at 30°C in a shaking incubator.

[0074] 5. Centrifuge at 1,500 g for 5 min at 4°C and remove the supernatant.

[0075] 6. Resuspend the cell pellet in 50 mL of SG / -Ura liquid medium and culture in a shaking incubator at 30°C.

[0076] 7. OD 600 The concentration was adjusted to 0.6, and after 10-fold serial dilution, the growth of yeast was evaluated on SD / -Ura Broth yeast-deficient solid medium containing different concentrations of NaCl (0 mM (denoted as control), 500 mM, and 1 M). The results are shown in Figure 3 , where A, C and E are solid culture medium results, and B, D and F are liquid culture medium results.

[0077] The results show that CtWRKY41 Overexpression had no significant effect on yeast growth ( Figure 3 Middle A). CtWRKY41Overexpressed yeast cells showed significant resistance in medium containing NaCl (500 mM and 1 M), especially when diluted 1000 times ( Figure 3 In addition, under liquid culture conditions (SD / -Ura Broth yeast defective liquid medium), the transformed CtWRKY41 After measuring the survival rate of yeast, it was found that under normal conditions without salt stress, the growth trends of the two yeast strains were basically the same ( Figure 3 Middle B). Under different concentrations of NaCl stress, CtWRKY41 The survival rate of the overexpression yeast strain was always higher than that of the control group. Specifically, under the stress levels of 500mM and 1M NaCl, after 9h of stress treatment, CtWRKY41 The survival rate of the overexpression yeast strain was significantly higher than that of the empty vector control strain ( Figure 3 These results indicate that CtWRKY41 enhances the yeast's ability to resist salt stress.

[0078] Example 3 CtWRKY41 Heterologous transformation of Arabidopsis thaliana and screening of positive plants

[0079] The method of Example 1 was used to construct CtWRKY41 The plant expression vector (i.e., 35S:CtWRKY41-eGFP) was transformed into Arabidopsis thaliana via the inflorescence dip method mediated by Agrobacterium GV3103. The wild-type Arabidopsis thaliana used in this study was the Columbia ecotype (Col-0). T0-generation transgenic plants were screened on 1 / 2 MS medium supplemented with 50 mg / L HYG. The T1 and T2 generations were subsequently screened using the same method to obtain homozygous transgenic lines.

[0080] To verify the transgenic plants, the homozygous CtWRKY41 The genomic DNA of transgenic Arabidopsis and wild-type Arabidopsis was used CtWRKY41 PCR amplification was performed using specific primers (CtWRKY41-eGFP-F and CtWRKY41-eGFP-R in Table 1 ), and the results were shown in Figure 1 . Figure 4 As shown, where +: CtWRKY41 Amplification product using plant expression vector as template; OE1-7: CtWRKY41 Transgenic lines: WT wild-type Arabidopsis thaliana. OE1, OE2, OE4, OE6, and OE7 were identified as positive plants.

[0081] At the same time, total RNA was extracted from homozygous transgenic Arabidopsis lines that were positive in both HYG screening and molecular identification, and qRT-PCR was used to detect the expression of WRKY41 in the transgenic lines using primers CtWRKY41-qPCR-F and CtWRKY41-qPCR-R in Table 1. CtWRKY41The expression levels of genes were measured. The primers for the internal reference genes were Atactin-F and Atactin-R in Table 1. Figure 5 , where ** indicates significant difference compared with WT ( p <0.01). It can be seen that OE1, OE2, OE4, OE6 and OE7 plants are successfully transformed CtWRKY41 Gene-positive plants provide reliable experimental materials for subsequent functional analysis.

[0082] Example 4 CtWRKY41 Involved in abscisic acid (ABA) response and enhanced salt tolerance in Arabidopsis

[0083] To determine CtWRKY41 Whether it is involved in the ABA signaling pathway, this example first analyzed the seeds screened in Example 3 through seed germination experiments. CtWRKY41 Sensitivity of transgenic lines to ABA treatment.

[0084] Mature T3 generation seeds and wild-type (WT) Arabidopsis seeds were collected, air-dried, and vernalized at 4°C for 2 days (carried out in a 4°C incubator). After vernalization, the seeds were treated with 70% ethanol for 10 minutes and then with anhydrous ethanol for 20 minutes to sterilize the surface. The sterilized Arabidopsis seeds were sown on 1 / 2MS solid culture medium (10cm×10cm square plate) and added with 150mM NaCl and 0.6μM ABA (denoted as ABA group). At the same time, a stress group with only 150mM NaCl added (denoted as NaCl group) and a control group without NaCl and ABA were set up. The three groups of seeds were placed in a growth chamber at 24°C, 16h light, 8h dark, and 70% relative humidity for germination and root length tests. Three parallel experiments were repeated for each group. The germination rate was recorded every 12 hours, and the root length was measured with a vernier caliper after 14 days. The results are shown in the table. Figure 6 and Figure 7 ,in, Figure 6 The middle left picture shows the results of the germination test, and the right picture shows the results of the root length test; Figure 7 ** indicates significant difference compared with WT ( p <0.01).

[0085] The results showed that treatment with 0.6 μM ABA significantly reduced CtWRKY41 The seed germination rates of transgenic lines and wild-type plants were significantly different, but the decrease in the germination rate of wild-type plants was more obvious. Under salt stress conditions, although NaCl treatment inhibited the germination rate of Arabidopsis 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, CtWRKY41The development of lateral roots of the overexpression (OE) strain was promoted. After 7 days of treatment with 0.6μM ABA, the main root length of the transgenic strain was significantly longer than that of the wild-type plant. After being treated with 150mM NaCl stress, although the root growth of both the wild-type and overexpression strains was inhibited, the degree of inhibition in the overexpression strain was significantly less. These results indicate that CtWRKY41 Overexpression of not only reduced the sensitivity of Arabidopsis to exogenous ABA, but also significantly enhanced the plant's resistance to salt stress. CtWRKY41 It plays an important role in integrating ABA signaling and salt stress response.

[0086] Example 5 CtWRKY41 Enhanced the physiological indicators and antioxidant capacity of transgenic Arabidopsis

[0087] To fully evaluate CtWRKY41 The effect of salt tolerance on Arabidopsis thaliana was studied in this example. CtWRKY41 The transgenic lines were subjected to salt stress treatment as follows:

[0088] Mature T3 seeds and wild-type (WT) Arabidopsis seeds were collected, air-dried, and vernalized at 4°C. Before sowing on 1 / 2 MS solid medium, seeds were surface-sterilized by treating with 70% ethanol for 10 minutes and then with anhydrous ethanol for 20 minutes. Sterilized seeds were sown on 1 / 2 MS medium and transferred to a growth chamber at 24°C with 16 hours of light and 8 hours of darkness, and a relative humidity of 70%. When four true leaves appeared, the plants were transplanted to a nutrient soil:vermiculite ratio of 1:3 (v / v). Fourteen days after transplanting, salt stress was applied by irrigating the plants with 150 mM NaCl solution. A control group was also maintained without salt stress. After 14 days of incubation, the plants were observed, their fresh weights recorded, and leaf samples were collected for histochemical staining with diaminobenzidine (DAB, brown) and nitro blue tetrazolium chloride (NBT, blue) to detect reactive oxygen species (ROS) accumulation and to measure chlorophyll content. See the 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).

[0089] 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 leaf dehydration and yellowing, but the degree of inhibition in the overexpression line was milder ( Figure 8 Middle A).

[0090] Under normal conditions, there was no significant difference in leaf staining between the wild type and the overexpression lines. However, under salt stress, the overexpression lines showed less blue and brown precipitates compared to the wild type ( Figure 8 B), which shows that its hydrogen peroxide (H2O2) and superoxide anion (O2 - ) accumulation was lower. 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).

[0091] These results indicate that CtWRKY41 The salt tolerance of transgenic Arabidopsis was significantly enhanced by improving antioxidant capacity and maintaining photosynthetic efficiency.

[0092] Salt stress usually leads to excessive accumulation of reactive oxygen species (ROS) in plants, causing severe oxidative damage. This example also uses a kit from Beijing Solebow Technology Co., Ltd. to detect the content of superoxide anion (H2O2), hydrogen peroxide (O2 - ) content, malondialdehyde (MDA) content, catalase (CAT) activity, superoxide dismutase (SOD) activity, peroxidase (POD) activity and proline (Pro) content of the culture medium. The results are shown in 9, where ** indicates significant differences compared with WT ( p <0.01).

[0093] The results showed that salt stress significantly increased the levels of malondialdehyde, superoxide anion, and hydrogen peroxide in Arabidopsis leaves. Compared with wild-type plants, the accumulation of malondialdehyde, superoxide anion, and hydrogen peroxide in the overexpression lines was significantly lower ( Figure 9 Salt stress significantly increased the activities of superoxide dismutase, catalase, and peroxidase in both the wild-type and overexpression lines, and also increased the accumulation of proline. Compared with the wild-type, the activities of these antioxidant enzymes and the content of proline in the overexpression lines were significantly higher ( Figure 9 (DG).

[0094] These results indicate that CtWRKY41 By enhancing antioxidant enzyme activity and osmotic regulation ability, the accumulation of ROS under salt stress conditions was effectively reduced, thereby alleviating oxidative damage and improving plant salt tolerance.

[0095] Example 6 CtWRKY41 Regulating the expression of stress-responsive genes

[0096] To clarify CtWRKY41 The molecular mechanism of enhancing salt tolerance in Arabidopsis thaliana was analyzed by RT-PCR using the primers in Table 1 to analyze the relative expression levels of stress response genes in wild type and overexpression strains under salt stress. Figure 10 .

[0097] The results showed that salt stress significantly induced the expression of multiple key genes in the overexpression lines. Compared with the wild type, the antioxidant enzyme-related genes ( AtCAT1 、 AtPOD1 and AtAPX1 ) were significantly more expressed. In addition, the expression of genes related to the abscisic acid signaling pathway ( AtNCED3 、 AtABI1 and AtABI2 ) was also significantly upregulated. Moreover, the expression level of the plasma membrane sodium antiporter gene ( AtSOS1 ) and dehydration-responsive genes ( AtRD22 ) was expressed at a significantly higher level in the overexpression line than in the wild type.

[0098] Example 7 CtWRKY41 Delaying flowering time in transgenic Arabidopsis

[0099] During the experiment, the inventors observed CtWRKY41 Involved in regulating the flowering time of Arabidopsis thaliana, this example systematically analyzes CtWRKY41 Effect on flowering time: The wild type and the CtWRKY41 The seeds of the overexpressing strains were vernalized and then cultured under 16 h light and 8 h dark conditions. The flowering time was recorded by observing the appearance of flower buds and the number of corresponding rosette leaves was counted. The wild type and CtWRKY41 The expression levels of several key flowering-related genes in the overexpression lines. Figure 11 , where A: phenotype of Arabidopsis thaliana, scale bar is 2 cm, B: number of rosette leaves at flowering, C: time of first flower opening in different strains, D: bolting time (i.e. time of appearance of inflorescence stem with height of 1 cm), E: overexpression CtWRKY41 Expression levels of endogenous flowering time-related genes in transgenic Arabidopsis thaliana; ** indicates significant differences compared with WT ( p <0.01).

[0100] The results showed that the wild-type plants had a bolting time of 30.5 days, a flowering time of 34.3 days, and a rosette leaf count of 12. CtWRKY41 The bolting time of the overexpression line was significantly delayed to 34 days, the flowering time was postponed to 37.8 days, and the number of rosette leaves increased to 17.5. These results indicate that CtWRKY41 Overexpression of CtWRKY41 Genes play an important role in integrating environmental signals and regulating flowering time.

[0101] Compared with the wild type, CtWRKY41Flowering integration genes in overexpression lines FT The expression of FLOWERING LOCUS T was significantly down-regulated, while the flowering repressor gene FLC The expression of FLOWERING LOCUS C was significantly upregulated. In addition, the key genes of the photoperiod pathway CO (CONSTANS) and key genes for gibberellin biosynthesis GA20OX The expression of gibberellin 20-oxidase was significantly upregulated in the CtWRKY41 overexpression lines, while the flowering integration gene SOC1 The expression of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 was significantly downregulated.

[0102] These results indicate that CtWRKY41 The expression of flowering-related genes can be synergistically regulated through multiple pathways. FLC and lower FT and SOC1 , CtWRKY41 The gene represses the flowering process. During the flowering transition, it is upregulated by CO and GA20OX to regulate the photoperiod and gibberellin pathways. This complex regulatory network reflects CtWRKY41 The discovery of WRKY transcription factors plays an important role in integrating different flowering signaling pathways, ultimately leading to a delayed flowering phenotype. This discovery provides new technical support for the role of WRKY transcription factors in the plant flowering regulatory network.

[0103] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

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

1.

2. A gene encoding the transcription factor CtWRKY41 of the radix cucurbitae according to 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: Comprising 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 CtWRKY41 transcription factor of claim 1 and / or the coding gene of claim 2 in one or more of the following: 1) Improve plant salt tolerance; 2) Delay plant flowering time; 3) Delay plant bolting time; 4) Reduce plant sensitivity to exogenous abscisic acid; 5) Increase the number of rosette leaves in plants; 6) Improve yeast salt tolerance; The plant is Arabidopsis thaliana.

9. 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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