Application of CpSnRK2.6 in regulation and control of stress resistance of cucurbita pepo

Abstract

The invention relates to the technical field of biology, in particular to application of CpSnRK2.6 to regulation and control of stress resistance of zucchini. It is found that CpSnRK2.6 can be induced by drought and salt stress, the transcriptional level of CpSnRK2.6 is rapidly increased after abscisic acid treatment, an overexpression plant is obtained by constructing an overexpression vector, a non-overexpressed plant has remarkable yellow leaves and wilting, a CpSnRK2.6 overexpressed plant only shows slight wilting symptoms, and the CpSnRK2.6 overexpressed plant has a good application prospect. The CpSnRK2.6 overexpressed plant relieves the leaf water loss and withering of the non-overexpressed plant, and after the CpSnRK2.6 overexpressed plant is rehydrated for 3 days, the CpSnRK2.6 overexpressed plant recovers normal growth, and the non-overexpressed plant cannot recover, so that the application of the CpSnRK2.6 in regulating and controlling the stress resistance of the zucchini is provided.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to application of CpSnRK2.6 in regulating the stress resistance of zucchini. Background Art

[0002] squash( Cucurbita pepo Zucchini (L.) belongs to the Cucurbitaceae family and is a genus that thrives in warm and humid climates. The young squash is primarily grown for consumption. Its thin skin and thick flesh are rich in nutrients like vitamin C and glucose. In recent years, zucchini cultivation has continued to expand with growing market demand, making it a key vegetable.

[0003] During the cultivation of zucchini, adverse climatic conditions such as high temperature and drought often occur, which have a significant impact on the physiological processes of plant seedlings, such as nutritional growth, photosynthesis, enzyme activity, and dry matter accumulation, thereby restricting the improvement of zucchini yield and quality. In order to cope with drought stress, the main existing technical means is to breed zucchini varieties with strong drought tolerance for cultivation. At present, the breeding of drought-resistant zucchini varieties mostly relies on conventional breeding methods. However, this method has a long breeding cycle, generally 5 to 8 years, or even longer, which makes it difficult to quickly meet the urgent demand for variety replacement in production. Therefore, there is an urgent need to provide a new strategy for zucchini to cope with drought stress. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a.

[0005] Application of CpSnRK2.6 in regulating stress resistance of zucchini.

[0006]

[0007] The amino acid sequence of CpSnrK2.6 is as follows: MDRSALTVGPGMDIPIMHDSDRYELVRDIGSGNFGIARLMRDKQTGELVAVKYIERGEKIDENVKREIINHRSLRHPNIVRFKEVILTPTHLAIVMEYASGGELFERICNAGRFSEDEARFFFQQLISGVSYCHAMQVCHRDLKLENTLLDGSPAPRLKICDFGYSKSSVLHSQPK STVGTPAYIAPEVLLKKEYDGKIADVWSCGVTLYVMLVGAYPFEDPEDPKNFRKTIQRILNVQYSIPDYVLISPECRHLISRIFVADPTKRITIPEIRNHEWFLKNLPADLVDERVMNNQYEEPDQPMQTSEEIMQIIAEAGLPAAGTQSLNQYLTGSLDIDDDMEDDLETDPDLDLDIDSSGEIVYAM, denoted as SEQ ID NO.2.

[0008] Preferably, the stress resistance is any one of drought resistance, salt stress resistance and hormone resistance.

[0009] Preferably, the regulation is to overexpress CpSnRK2.6 to improve the drought resistance of zucchini.

[0010] Preferably, the regulation is to overexpress CpSnRK2.6 to improve the ability of zucchini to resist salt stress.

[0011] Preferably, the method for regulating the stress resistance of zucchini is: obtaining transgenic zucchini by overexpressing the CpSnRK2.6 to regulate the stress resistance of zucchini.

[0012] Preferably, the method for overexpressing CpSnRK2.6 is: replacing the eGFP sequence in the ZYMV-eGFP viral vector with the CDS of CpSnRK2.6 to construct a ZYMV-CpSnRK2.6 recombinant plasmid to overexpress CpSnRK2.6.

[0013] Preferably, sequence replacement is performed at the ApaI / SalI restriction site.

[0014] Preferably, the nucleotide sequences of the primers for detecting the expression of CpSnRK2.6 are as shown in SEQ ID NO.33 to SEQ ID NO.34.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention discovered that CpSnRK2.6 can be induced by drought and salt stress, and its transcription level rapidly increases after abscisic acid treatment. By constructing an overexpression vector, overexpression plants were obtained. Non-overexpression plants showed significant yellowing and wilting of leaves, while CpSnRK2.6-overexpressing plants only showed mild wilting symptoms. In addition, CpSnRK2.6-overexpressing plants alleviated leaf water loss and wilting of non-overexpression plants. After rehydration for 3 days, CpSnRK2.6-overexpressing plants resumed normal growth, while non-overexpression plants failed to recover. Therefore, the application of CpSnRK2.6 in regulating the stress resistance of zucchini is proposed. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Expression pattern analysis of CpSnRK2.6; A: expression level under drought and salt stress treatment; B: expression level under abscisic acid treatment.

[0017] Figure 2 This is a multiple sequence alignment of CpSnRK2.6 protein, among which digsgnfga in the Consensus of the first group is the ATP binding site, chrdlklentll in the Consensus of the second group is the kinase domain, and Ididddmedledddldidssgeivya in the Consensus of the fifth group is the ABA response element.

[0018] Figure 3 The subcellular localization of the CpSnRK2.6 protein is shown. Blue shading indicates the degree of sequence homology, based on percent sequence identity in ClustalOmega. Structure-based sequence alignment identified multiple conserved motifs and domains, including the ATP-binding loop, ATP-binding site, kinase domain, and abscisic acid box. Green fluorescent protein (GFP)-tagged CpSnRK2.6 was transiently expressed in epidermal cells of Nicotiana benthamiana plants using Agrobacterium infiltration. Markers: pBI221-NLS-CFP, pBI221-mCherry-PM. Scale bar represents 20 μm.

[0019] Figure 4 To obtain CpSnRK2.6 overexpressing plants, A: Symptom one of zucchini plants 12 days after inoculation with ZYMV infectious clones; B: Symptom two of zucchini plants 12 days after inoculation with ZYMV infectious clones; C: Fluorescence phenotype one of zucchini plants 12 days after inoculation with ZYMV infectious clones; D: Fluorescence phenotype two of zucchini plants 12 days after inoculation with ZYMV infectious clones.

[0020] Figure 5 qPCR analysis of zucchini CpSnRK2.6 overexpressing plants.

[0021] Figure 6 To enhance the resistance of zucchini to salt stress and drought stress by overexpressing CpSnRK2.6; A: seedling growth of ZYMV-eGFP strain under salt stress; B: seedling growth of ZYMV-CpSnRK2.6 strain under salt stress; C: seedling growth of ZYMV-eGFP strain under drought stress; D: seedling growth of ZYMV-CpSnRK2.6 strain under drought stress; E: seedling growth of ZYMV-eGFP strain under rehydration; F: seedling growth of ZYMV-CpSnRK2.6 strain under rehydration; A(1)~F(1) are the fluorescence phenotypic characteristics of seedlings A~F under handheld UV lamp irradiation, respectively.

[0022] Figure 7 Overexpression of CpSnRK2.6 enhances the antioxidant activity of zucchini; A: DAB brown and NBT blue staining; B-H: Analysis of physiological indicators under salt stress, where B is malondialdehyde content, C is hydrogen peroxide content, D is superoxide anion content, E is proline content, F is superoxide dismutase activity, G is peroxidase activity, and H is catalase activity. Scale bar = 2 mm.

[0023] Figure 8 Stomatal closure characteristics of CpSnRK2.6 transgenic lines under different treatment conditions.

[0024] Figure 9 Chlorophyll content and fluorescence parameters of CpSnRK2.6 transgenic lines under salt stress and drought stress; A: Chlorophyll content; B: Photosystem II quantum yield; C: Non-photochemical quenching.

[0025] Figure 10 The interaction network of CpPP2Cs-CpSnRK proteins.

[0026] Figure 11 Interaction analysis of CpSnRK2.6 with CpPP2C1 and CpPP2C4; A: Yeast two-hybrid assay showed that CpSnRK2.6 interacts with CpPP2C1 and CpPP2C4; B: Bimolecular fluorescence complementation assay verified the interaction of CpSnRK2.6 with CpPP2C1 and CpPP2C4 in tobacco leaves; yeast strains were evaluated using a 1,1 / 10 serial dilution method; Scale bar = 20 μm.

[0027] Figure 12Interaction analysis between CpPYL / PYRs and CpPP2C; A: yeast two-hybrid interaction between CpPP2C1 and CpPYL / PYRs; B: yeast two-hybrid interaction between CpPP2C4 and CpPYL / PYRs; yeast strain AH109 was transformed with different plasmid combinations and cultured on selective media: SD / -Leu / -Trp, SD / -Leu / -Trp / -Ade / -His, and SD / -Leu / -Trp / -Ade / -His / +10μM ABA. Yeast strains were evaluated using a 1, 1 / 10 serial dilution method. DETAILED DESCRIPTION

[0028] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified.

[0029] Double-deficient medium: SD / -Leu / -Trp is SD medium without leucine and tryptophan.

[0030] Four-deficient medium: SD / -Leu / -Trp / -Ade / -His, which is SD medium without leucine, tryptophan, adenine and histidine.

[0031] Four ABA-deficient culture media: SD / -Leu / -Trp / -Ade / -His / +10 μM ABA, which is SD culture media without leucine, tryptophan, adenine, and histidine, and the ABA concentration in the SD culture media is 10 μM.

[0032] 1. Materials The present invention uses zucchini ( C. pepo ) homozygous inbred line "YF." Seeds were sown in a 1:1 mixture of soil and sand and cultured in a controlled climate chamber with a day / night temperature of 28 / 22°C and a photoperiod of 18 / 6 hours light / dark. "YF" refers to the reference "S.H. Li, P. Wang. Increasing forms overexpression of CpVQ30 increases susceptibility of Cucurbita pepo topowdery mildew via oxidative stress and callose degradation." Scientia Horticulturae, 2025.

[0033] KpnI and XbaI restriction enzymes were purchased from TaKaRa, China. ApaI / SalI restriction sites were purchased from TaKaRa, China. Commercial detection kits were purchased from Solarbio, China. DAB and NBT solutions were purchased from Solarbio, China.

[0034] The primer sequences used in the present invention are shown in Table 1.

[0035] Table 1 Primer sequences of related genes

[0036] 2. Zucchini seedling stress treatment and sample collection When the seedlings grew to the two-leaf and one-heart stage, the following stress treatments were carried out: (1) ABA treatment: ABA was dissolved in a small amount of 95% ethanol, diluted with distilled water to a working concentration of 100 μM, and evenly sprayed on the leaves of the seedlings. Leaf samples were collected at 0, 1, 3, 6, 9, and 12 hours after treatment, immediately frozen in liquid nitrogen, and stored at -80°C for RNA extraction. The treatment time 0 was used as a control.

[0037] (2) PEG 6000 drought stress simulation: A 20% PEG 6000 aqueous solution was used to simulate drought conditions. The seedling roots were completely immersed in the treatment solution. Samples were taken at 0, 6, 12, and 24 hours, quickly frozen in liquid nitrogen, and stored for later use. The treatment time 0 was used as the control.

[0038] (3) NaCl salt stress treatment: Prepare a 200 mM NaCl aqueous solution and immerse the seedling roots completely in it for salt stress treatment. Samples were taken at 0, 6, 12, and 24 hours, quickly frozen in liquid nitrogen, and stored for later use. The treatment time 0 was used as the control.

[0039] 3. CpSnRK2 gene expression analysis RNA extraction and gene expression analysis were performed according to the literature Xu, K., Wang, P. (2024). Transcriptome-wide identification of the Hsp70 gene family in Pugionium cornutum and functional analysis of PcHsp70-5 under drought stress. Planta, 260(4), 84. ACT and CAC genes were used as internal references. The relative expression of target genes was calculated using 2 -ΔΔCT All experiments were repeated at least 3 times.

[0040] 4. Subcellular localization and virus-induced gene overexpression Subcellular localization experiments were performed using the pCAMBIA1300-35S-eGFP vector system. The vector was linearized using KpnI and XbaI restriction endonucleases, and the CpSnRK2.6 coding sequence CDS was inserted into the KpnI / XbaI sites using seamless cloning. The recombinant plasmid was transformed into Agrobacterium, and subcellular localization analysis was performed according to Xu, K., Wang, P. (2024). Transcriptome-wide identification of the Hsp70 gene family in Pugionium cornutum and functional analysis of PcHsp70-5 under drought stress. Planta, 260(4), 84.

[0041] Transient overexpression analysis used the ZYMV-eGFP viral vector system. The ZYMV-eGFP viral vector is described in Liu Liming, Kang Baoshan, Peng Bin, et al. Construction of a ZYMV infectious clone carrying eGFP and its infectivity [J]. Acta Phytopathologica Sinica, 2021, 51(05):734-740. Using seamless cloning technology, the eGFP sequence in the vector was replaced with the CpSnRK2.6 CDS at the ApaI / SalI restriction site to construct the ZYMV-CpSnRK2.6 recombinant plasmid. The ZYMV-eGFP (control) and ZYMV-CpSnRK2.6 plasmids were transformed into Agrobacterium tumefaciens GV3101 strain by freeze-thaw method. The bacterial solution concentration was adjusted to OD600 = 1.0, and the fully expanded cotyledons of healthy zucchini seedlings were infected with a sterile syringe. ≥10 seedlings were used for each treatment. The inoculated plants were cultured at 28 ± 1°C with a 16-h light / 8-h dark photoperiod.

[0042] 5. Determination of physiological and biochemical indicators The contents of malondialdehyde, proline, superoxide anion, and hydrogen peroxide, as well as the activities of superoxide dismutase, peroxidase, and catalase, were measured using commercially available test kits according to the manufacturer's instructions. Histochemical detection of reactive oxygen species was performed using 3,3'-diaminobenzidine and nitroblue tetrazolium staining, where malondialdehyde is abbreviated as MDA, proline as Pro, and superoxide anion as O2 - , hydrogen peroxide is abbreviated as H2O2, superoxide dismutase is abbreviated as SOD, peroxidase is abbreviated as POD, catalase is abbreviated as CAT, reactive oxygen species is abbreviated as ROS, 3,3'-diaminobenzidine is abbreviated as DAB, and nitroblue tetrazolium is abbreviated as NBT.

[0043] The brief steps were as follows: fresh leaves of stress-treated and control plants were immersed in DAB and NBT solutions, respectively, and incubated in the dark at room temperature. The staining procedure followed the method in the literature Sekulska-Nalewajko J, Gocławski J, Chojak-KoźniewskaJ, Kuźniak E (2016) Automated image analysis for quantification of reactive oxygen species in plant leaves. Methods (San Diego, Calif.) 109: 114–122.doi:10.1016 / j.ymeth.2016.05.018.

[0044] To investigate the role of CpSnRK2.6 in responses to salt and drought stress, wild-type WT and CpSnRK2.6-overexpressing plants were subjected to stress treatment. Stomatal aperture dynamics in the lower epidermis of detached leaves were observed microscopically. Chlorophyll content was determined using 95% ethanol extraction, and chlorophyll fluorescence parameters, including the nonphotochemical quenching coefficient (NPQ) and the photosystem II quantum yield (ΦPSII), were measured using a LI-6400 portable photosynthesis system.

[0045] 6. Yeast two-hybrid and bimolecular fluorescence complementation experiments Yeast two-hybrid was abbreviated as Y2H: the CDSs of CpSnRK2.6, CpPP2C1, and CpPP2C4 were cloned into the pGBKT7 vector, and the CDSs of CpPP2Cs and CpPYL / PYRs were inserted into the pGADT7 vector.

[0046] Bimolecular fluorescence complementation is abbreviated as BiFC: the CpSnRK2.6 CDS was fused to the 35S:nYFP vector, and the CpPP2C1 and CpPP2C4 CDS were cloned into the 35S:cYFP vector.

[0047] Both Y2H and BiFC experiments were performed as reported in the literature Liu, Y., Wu, G., Zhao, Y., Wang, HH, Dai, Z., Xue, W., Yang, J., Wei, H., Shen, R.,&Wang, H. (2021). DWARF53 interacts with transcription factors UB2 / UB3 / TSH4 to regulate maize tillering and tasselbranching. Plant physiology, 187(2), 947–962.

[0048] 7. Statistical analysis Statistical analysis was performed using Student's t test based on experimental data. Asterisks indicate significant differences from three biological replicates, *p < 0.05 and **p < 0.01.

[0049] result 1. Analysis of CpSnRK2.6 expression pattern like Figure 1 As shown in Figure 2, CpSnRK2.6 can be induced by drought and salt stress, and its transcription level increases rapidly after abscisic acid treatment. Compared with 0h, the expression of CpSnRK2.6 under salt stress reaches a peak at 6h and then gradually decreases, but is still higher than the control, as shown in Figure 2. Figure 1 A. Compared with 0h, the expression of CpSnRK2.6 gradually increased after drought stress and reached a peak at 24h. Figure 1 B. Under ABA treatment, the transcription level of CpSnRK2.6 increased sharply within 1 hour and reached a peak at 3 hours. Figure 1 These results indicate that CpSnRK2.6 responds to drought, salt stress, and ABA treatment, suggesting that it may be involved in ABA-mediated stress resistance regulation in zucchini.

[0050] 2. Sequence alignment and subcellular localization of CpSnRK2.6 Based on preliminary expression analysis, CpSnRK2.6 showed significant responses to various stresses, including ABA, PEG, and NaCl, and was therefore selected as a key research target. Its full-length 1098bp CDS sequence was successfully cloned from the zucchini inbred line MRJ, encoding a 365-amino acid protein kinase. Multiple sequence alignment revealed the presence of a highly conserved Domain I at its C-terminus. Domain II specifically refers to the ABA response element in the SnRK2 gene, and is similar in sequence to the corresponding domains in AtSnRK2.6, such as Figure 2This conserved domain has a key function. Studies have shown that it is directly involved in mediating the interaction with protein phosphatase 2Cs, thereby regulating the ABA signaling pathway. Protein phosphatase 2Cs is abbreviated as PP2Cs.

[0051] To explore the subcellular localization of CpSnRK2.6, a 35S::CpSnRK2.6-eGFP fusion expression vector was constructed and transiently expressed in tobacco epidermal cells by Agrobacterium-mediated transformation. Experimental controls included a negative control, a nuclear marker, and a plasma membrane marker, which were labeled pCAMBIA1300-eGFP, pBI221-NLS-CFP, and pBI221-mCherry-PM, respectively. Confocal microscopy analysis showed that CpSnRK2.6-GFP fluorescence was mainly localized in the nucleus and plasma membrane, and showed clear co-localization with the NLS-CFP nuclear marker and the mCherry-PM plasma membrane marker. These results confirmed that CpSnRK2.6 has dual localization characteristics in the nucleus and plasma membrane in plant cells, such as Figure 3 .

[0052] 3. ZYMV-mediated transient overexpression of CpSnRK2.6 enhances drought and salt stress tolerance in zucchini. ZYMV is zucchini yellow mosaic virus.

[0053] The ZYMV-CpSnRK2.6 plasmid was introduced into Agrobacterium tumefaciens GV3101 strain, and Agrobacterium infected with ZYMV-eGFP was used as a positive control for Agrobacterium-mediated transformation of zucchini plants. On the 12th day after inoculation, the new leaves of all plants showed obvious mosaic deformity symptoms, such as Figure 4 Under a handheld UV lamp, the ZYMV-CpSnRK2.6-inoculated plants showed no fluorescence, while the leaves of the ZYMV-eGFP-inoculated plants showed strong fluorescence. qPCR analysis confirmed that the expression level of CpSnRK2.6 in the ZYMV-CpSnRK2.6-inoculated plants was significantly higher than that in the ZYMV-eGFP control group. Figure 5 .

[0054] To evaluate the effects of CpSnRK2.6 overexpression under salt stress and drought stress, the docked plants were subjected to salt stress and natural drought treatments. After 7 days of salt stress, ZYMV-eGFP plants showed significant yellowing and wilting, while ZYMV-CpSnRK2.6 overexpression plants showed only mild wilting symptoms, such as Figure 6 A and B in Figure 2. After 7 days of drought stress, both ZYMV-eGFP and ZYMV-CpSnRK2.6 plants experienced leaf dehydration and wilting, but the symptoms of ZYMV-eGFP plants were more severe. Figure 6However, 3 days after rehydration, ZYMV-CpSnRK2.6 plants resumed normal growth, while ZYMV-eGFP plants failed to recover, as shown in Figure 4. Figure 6 E and F in .

[0055] 4. Overexpression of CpSnRK2.6 enhances the antioxidant capacity of zucchini, promotes photosynthesis and regulates stomatal closure.

[0056] Salt stress and drought stress usually induce oxidative damage in plants. In the present invention, both stress treatments resulted in the increase of MDA and O2 in zucchini leaves. - NBT blue and DAB brown staining showed that the blue and brown staining intensities of the leaves of ZYMV-CpSnRK2.6 plants under salt stress were weaker than those of the ZYMV-eGFP control plants, indicating that the H2O2 and O2 - Less accumulation, such as Figure 7 A. Quantitative analysis confirmed that MDA, O2 - and H2O2 levels were significantly lower than those in the ZYMV-eGFP control group. Figure 7 Antioxidant enzyme activity analysis showed that salt stress and drought stress could induce the increase of SOD, CAT, POD activities and proline content in ZYMV-CpSnRK2.6 and ZYMV-eGFP plants. Figure 7 Notably, ZYMV-CpSnRK2.6 plants displayed significantly higher SOD, CAT, and POD activities, as well as greater proline accumulation. These results suggest that CpSnRK2.6 mitigates oxidative damage under salt stress by enhancing antioxidant defense mechanisms, thereby reducing ROS accumulation.

[0057] Under salt stress and drought stress conditions, the stomatal movement of CpSnRK2.6 transgenic plants was significantly different from that of ZYMV-eGFP control plants: the stomatal closure speed of transgenic plants was faster, as shown in Figure 2. Figure 8 This suggests that overexpression of CpSnRK2.6 enhances the plant's response and tolerance to stress by promoting stomatal closure and reducing water loss under osmotic stress.

[0058] In terms of photosynthetic performance, there was no significant difference in chlorophyll content and fluorescence parameters between CpSnRK2.6 transgenic plants and ZYMV-eGFP control plants under normal growth conditions. However, under salt stress and drought stress, CpSnRK2.6 transgenic plants showed obvious photosynthetic advantages: the chlorophyll content and ФPSII of transgenic plants were significantly higher, while NPQ was significantly lower than that of ZYMV-eGFP control plants, such as Figure 9These results indicate that CpSnRK2.6 maintains photosynthetic carbon assimilation under osmotic stress by enhancing the plant's ability to absorb, transmit, and convert light energy. This physiological advantage is crucial for dry matter production under stress conditions and provides a solid foundation for improving the stress resistance of CpSnRK2.6 transgenic lines.

[0059] 5. Interaction analysis of CpPYLs-CpPP2Cs-CpSnRK2.6 In the ABA signaling pathway, class A PP2C proteins act as key negative regulators by directly binding to and inhibiting the kinase activity of SnRK2. To explore the interaction between CpSnRK2.6 and class A PP2C proteins in zucchini, a protein-protein interaction network was constructed using the STRING database. Prediction analysis showed that there were potential interactions between six CpSnRK2 proteins and seven PP2C family members, such as Figure 10 However, no interaction signals were detected between CpSnRK2 proteins themselves.

[0060] To verify the above prediction, five class A PP2C proteins, designated PP2C1, PP2C2, PP2C3, PP2C4, and PP2C5, were selected for Y2H analysis with CpSnRK2.6. Only the combinations AD-PP2C1 / BD-CpSnRK2.6 and AD-PP2C4 / BD-CpSnRK2.6 could grow normally in the four-deficient medium, designated SD / -Ade / -His / -Leu / -Trp, indicating that CpSnRK2.6 interacts specifically with PP2C1 / PP2C4. Figure 11 A. BiFC experiments further confirmed this result: in tobacco epidermal cells co-expressing nYFP-PP2C1 / cYFP-CpSnRK2.6 and nYFP-PP2C4 / cYFP-CpSnRK2.6, obvious YFP fluorescence signals were observed, as shown in Figure 11 B. These results provide key evidence for the molecular mechanism of ABA signal transduction in zucchini, indicating that CpSnRK2.6 may regulate the ABA signaling pathway through specific interactions with PP2C1 and PP2C4.

[0061] To investigate the interaction between CpPP2Cs and the ABA receptors CpPYL / PYRs, we cloned CpPYL / PYRs genes from zucchini and analyzed them using a Y2H assay. Under the conditions of SD / -Leu / -Trp / -Ade / -His, CpPP2C1 interacted only with CpPYL1. However, after adding 10 μM ABA, CpPP2C1 interacted with CpPYL1, CpPYL2, CpPYL3, and CpPYR2, as shown in Figure 2. Figure 12Similarly, under ABA-free conditions, CpPP2C4 interacted only with CpPYL1 and CpPYL2, but after ABA treatment, its interaction range was extended to all tested CpPYL / PYRs, such as Figure 12 These results indicate that ABA can enhance the interaction network between CpPP2Cs and CpPYL / PYRs.

[0062] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes preferred embodiments.

[0063] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0064] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. Application of CpSnRK2.6 in regulating stress resistance of zucchini, characterized in that: The nucleotide sequence of CDS of CpSnRK2.6 is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The stress resistance is any one of drought resistance, salt stress resistance and hormone resistance.

3. The use according to claim 2, characterized in that The regulation is to overexpress CpSnRK2.6 to improve the drought resistance of zucchini.

4. The use according to claim 2, characterized in that The regulation is to overexpress CpSnRK2.6 to improve the ability of zucchini to resist salt stress.

5. The use according to claim 1, characterized in that The method for regulating the stress resistance of zucchini is: obtaining transgenic zucchini by overexpressing the CpSnRK2.6, so as to regulate the stress resistance of zucchini.

6. The use according to claim 5, characterized in that The method for overexpressing CpSnRK2.6 is as follows: the eGFP sequence in the ZYMV-eGFP viral vector is replaced with the CDS of CpSnRK2.6 to construct a ZYMV-CpSnRK2.6 recombinant plasmid to overexpress CpSnRK2.

6.

7. The use according to claim 6, characterized in that Sequence replacement was performed at the ApaI / SalI restriction site.

8. The use according to claim 6, characterized in that The nucleotide sequences of the primers for detecting the expression of CpSnRK2.6 are shown in SEQ ID NO.33 to SEQ ID NO.34.

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