Application of CpJAZ13 and its recombinant overexpression vector in improving drought stress resistance in zucchini

Abstract

The present invention belongs to the field of biological genetic engineering technology and specifically relates to a CpJAZ13 and application of a recombinant overexpression vector thereof in improving the drought stress resistance of zucchini, CpJAZ13 The nucleotide sequence of is shown in SEQ ID NO. 1. The present invention is the first to achieve the overexpression system in zucchini mediated by ZYMV virus. CpJAZ13 and confirmed its positive effect on drought resistance in zucchini. CpJAZ13 The expression is upregulated after drought stress treatment. CpJAZ13 Improved drought resistance in yeast; transient overexpression CpJAZ13 The antioxidant capacity of zucchini is increased, including reduced ROS accumulation and enhanced antioxidant enzyme activity, thereby improving zucchini's resistance to drought stress. Compared to the lengthy breeding cycle of traditional hybridization, this invention can shorten the breeding time to just a few months. This invention provides a new genetic resource for zucchini variety improvement and can promote the development of high-quality, drought-resistant zucchini varieties to meet agricultural production and market demands.

Description

Technical Field

[0001] The present invention belongs to the field of biological genetic engineering technology and specifically relates to a CpJAZ13 and application of its recombinant overexpression vector in improving zucchini's resistance to drought stress. 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 negative impact of drought stress on zucchini, the present invention provides a CpJAZ13 Application in improving zucchini's resistance to drought stress.

[0005] The technical solution adopted in the present invention is:

[0006] A first aspect of the present invention provides a CpJAZ13 Application in improving the drought stress resistance of zucchini, CpJAZ13 The nucleotide sequence is shown in SEQ ID NO.1.

[0007] A second aspect of the present invention provides a CpJAZ13 recombinant overexpression vector.

[0008] The third aspect of the present invention provides a method for preparing the recombinant overexpression vector, comprising the following steps:

[0009] Total RNA of wild-type zucchini was extracted and reverse transcribed into cDNA. Using cDNA as template, PCR amplification was performed to obtain CpJAZ13 After enzyme digestion of the expression vector, seamless cloning was used to CpJAZ13 The recombinant overexpression vector is obtained by connecting it to the expression vector after enzyme digestion.

[0010] Preferably, the expression vector is pXT1-ZYMV-eGFP.

[0011] Preferably, the restriction endonuclease used to cleave the expression vector is Apa I and Sale I.

[0012] A fourth aspect of the present invention provides an application of the recombinant overexpression vector, wherein the recombinant overexpression vector is used to construct an overexpression plant to improve the resistance of zucchini to drought stress.

[0013] Preferably, the method for constructing the overexpression plant comprises the following steps:

[0014] The recombinant overexpression vector is transferred into Agrobacterium competent cells, cultured amplified, and an infection solution is prepared; the infection solution is used to infect zucchini and then cultured to obtain overexpression plants.

[0015] Preferably, the Agrobacterium competent cell is GV3101.

[0016] Preferably, the infected part of the zucchini is the cotyledon.

[0017] Preferably, the expanded culture is carried out at 28° C. for 16 hours.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention provides a CpJAZ13 Application in improving the drought stress resistance of zucchini, CpJAZ13 The nucleotide sequence of is shown in SEQ ID NO. 1. The present invention is the first to achieve the overexpression system in zucchini mediated by ZYMV virus. CpJAZ13 and confirmed its positive effect on drought resistance in zucchini. CpJAZ13 The expression is upregulated after drought stress treatment. CpJAZ13 Improved drought resistance in yeast; transient overexpression CpJAZ13 The antioxidant capacity of zucchini is increased, including reduced ROS accumulation and enhanced antioxidant enzyme activity, thereby improving zucchini's resistance to drought stress. Compared to the lengthy breeding cycle of traditional hybridization, this invention can shorten the breeding time to just a few months. This invention provides a new genetic resource for zucchini variety improvement and can promote the development of high-quality, drought-resistant zucchini varieties to meet agricultural production and market demands.

[0020] In addition, the present invention deeply explores the functional genes related to drought stress response and explains its molecular mechanism of action, which is of great significance for the creation of drought-resistant zucchini germplasm resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Under drought stress with PEG6000 containing 20% ​​mass fraction CpJAZ13 expression of the situation.

[0022] Figure 2 Tobacco leaves CpJAZ13 Subcellular localization analysis of pBI121-eGFP, A~D are respectively: pBI121-eGFP signals in GFP channel, mCherry-Nucleus channel, Merge channel and DIC; E~H are respectively: pBI121- CpJAZ13 -eGFP signals in GFP channel, mCherry-Nucleus channel, Merge channel, and DIC.

[0023] Figure 3 For overexpression CpJAZ13 Improved the resistance of yeast to drought stress. A~E are: Without drought treatment, yeast containing pYES2 empty vector showed 0 , 10 -1 , 10 -2 , 10 -3 and 10 -4 The growth status of yeast when a~e are: without drought treatment, containing pYES2- CpJAZ13 The yeast of the carrier was instilled for 10 0 , 10 -1 , 10 -2 , 10 -3 and 10 -4 The growth status of yeast at 10 0 , 10 -1 , 10 -2 , 10 -3 and 10 -4 The growth status of yeast at the time of treatment; f~j are: when treated with 5% PEG6000, the yeast containing pYES2- CpJAZ13 The yeast of the carrier was instilled for 10 0 , 10 -1 , 10 -2 , 10 -3 and 10 -4 The growth status of yeast at 10% mass fraction of PEG6000; K~O are: when the mass fraction of PEG6000 is 10%, the yeast containing pYES2 empty vector is 0 , 10 -1 , 10 -2 , 10 -3 and 10 -4The growth status of yeast at the time of treatment; k~o are: when the mass fraction of PEG6000 is 10%, the yeast containing pYES2- CpJAZ13 Yeast instillation of carrier 10 0 , 10 -1 , 10 -2 , 10 -3 and 10 -4 Yeast growth condition.

[0024] Figure 4 For overexpression CpJAZ13 Improved the resistance of zucchini to drought stress. A~C are the front view, top view and fluorescence detection images of zucchini after ZYMV-eGFP infection; D~F are the ZYMV- CpJAZ13 Front view, top view and fluorescence detection image of zucchini after infection; G~I are: phenotypes of zucchini infected with ZYMV-eGFP after 4 days of drought stress, 7 days of drought stress and 2 days of rewatering; J~L are: ZYMV- CpJAZ13 Phenotypes of zucchini after 4 days of drought stress, 7 days of drought stress and 2 days of rewatering after infection.

[0025] Figure 5 For overexpression CpJAZ13 PCR detection of zucchini plants.

[0026] Figure 6 For overexpression CpJAZ13 Expression analysis of zucchini plants.

[0027] Figure 7 Results of DAB and NBT staining. A: Overexpression CpJAZ13 NBT staining of three parallel samples of plant leaves; B: overexpression CpJAZ13 A: DAB staining of three parallel samples of plant leaves; C: NBT staining of three parallel samples of control plants; D: DAB staining of three parallel samples of control plants.

[0028] Figure 8 For overexpression CpJAZ13 The results of antioxidant enzyme activity test of zucchini plants, A~F are H2O2 content, O2 - content, MDA content, POD content, SOD content and CAT content. DETAILED DESCRIPTION

[0029] The present invention will be further described below by way of specific examples, but the scope of the present invention is not limited thereto. The details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but such modifications or replacements fall within the scope of protection of the present invention.

[0030] The inventive concept of the present invention is as follows:

[0031] During zucchini cultivation, adverse climatic conditions such as high temperature and drought significantly impact physiological processes such as vegetative growth, photosynthesis, enzyme activity, and dry matter accumulation in young plants, thereby restricting the yield and quality of zucchini. To address drought stress, the main existing technical approach is to cultivate drought-tolerant zucchini varieties. Currently, the selection and breeding of drought-tolerant zucchini varieties mostly relies on conventional breeding methods. However, this method has a lengthy breeding cycle, typically 5 to 8 years or even longer, making it difficult to quickly meet the urgent demand for variety replacement in production.

[0032] Based on this, the present invention provides a CpJAZ13 Application in improving the drought stress resistance of zucchini, CpJAZ13 The nucleotide sequence is shown in SEQ ID NO.1.

[0033] In order to make those skilled in the art better understand the technical solution of the present invention and be able to implement it, the present invention is further described below in conjunction with specific examples. In the description of the present invention, unless otherwise specified, the reagents used are all commercially available and the methods used are all conventional techniques in the art.

[0034] The abbreviations of the present invention are shown in Table 1.

[0035] Table 1 Abbreviations

[0036]

[0037] Example 1

[0038] CpJAZ13 The application of the recombinant overexpression vector thereof in improving the drought stress resistance of zucchini is as follows:

[0039] 1. Materials and methods

[0040] (1) Plant materials and treatments.

[0041] The zucchini used in the present invention is a self-pollinating zucchini. Zucchini seeds are planted in a mixture of soil and vermiculite in a mass ratio of 1:1 and cultured in an artificial climate chamber with a photoperiod of 18h / 6h and 28°C / 22°C. After 8 weeks, the germinated zucchini seedlings are placed under drought stress conditions for co-culture for 24h, and leaves are collected at 0h, 6h, 12h and 24h, respectively. The treatment condition for drought stress is PEG6000 with a mass fraction of 20%. After collecting the leaves, all the leaves are immediately frozen in liquid nitrogen and stored at -80°C for extraction of total RNA. All leaves are tested using 3 technical replicates and 3 independent biological replicates.

[0042] (2) CpJAZ13 Gene expression analysis.

[0043] RNA extraction and CpJAZ13 Reference for expression analysis: Xu, K.,&Wang, P. (2024). Transcriptome-wide identification of the Hsp70 gene family in Pugioniumcornutum and functional analysis of PcHsp70-5 under drought stress. Planta,260(4), 84. https: / / doi.org / 10.1007 / s00425-024-04509-9. actin and CAC gene as an internal reference gene. CpJAZ13 The relative expression level was 2 -ΔΔCT All experiments were repeated at least three times. The primers used in the present invention are listed in Table 2.

[0044] Table 2 Primers used in the present invention

[0045]

[0046] Note: Since the present invention adopts homologous recombination to carry out gene cloning, the lowercase letters in the sequence information in Table 2 represent homology arms, and the uppercase letters represent the 5' end and 3' end of the corresponding gene.

[0047] (3) Construction of recombinant vector.

[0048] CpJAZ13 The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. The following vector construction process uses the vector shown in SEQ ID NO.1. CpJAZ13 nucleotide sequence.

[0049] 1) Use the pBI121-eGFP vector to construct a vector for subcellular localization as follows:

[0050] pBI121-eGFP vector was Xba I and Bam After HI digestion, the primers shown in SEQ ID NO.11 and SEQ ID NO.12 were used to clone the CpJAZ13 cloned into pBI121-eGFP vector Bam HI and Xba I site in the middle, the obtained pBI121- CpJAZ13-eGFP vector was injected into tobacco leaves for subcellular localization.

[0051] 2) Use pYES2 vector to express CpJAZ13 Perform overexpression experiments as follows:

[0052] pYES2 vector Bam HI and Eco After RI digestion, the primers shown in SEQ ID NO.5 and SEQ ID NO.6 were used to clone the CpJAZ13 Clone into pYES2 vector to obtain pYES2- CpJAZ13 Then, the pYES2- CpJAZ13 The vector and the empty pYES2 vector were transformed into the Saccharomyces cerevisiae strain INVScl. After incubation at 30°C for 40 h, positive clones were screened on SD-U medium. Yeast growth was assessed by dropping 10-fold serial dilutions onto solid SD-U medium.

[0053] 3) Using the expression vector pXT1-ZYMV-eGFP in zucchini CpJAZ13 Perform overexpression experiments as follows:

[0054] Total RNA of wild-type zucchini was extracted and reverse transcribed into cDNA. Using cDNA as template, PCR amplification was performed to obtain CpJAZ13 The expression vector pXT1-ZYMV-eGFP was Apa I and Sale After enzyme digestion with SEQ ID NO.9 and SEQ ID NO.10, the primers were used to clone the DNA fragments of the SEQ ID NO.9 and SEQ ID NO.10 by seamless cloning technology. CpJAZ13 Clone into pXT1-ZYMV-eGFP vector to obtain the recombinant overexpression vector pXT1-ZYMV- CpJAZ13 For ease of explanation, in the following text, pXT1-ZYMV- CpJAZ13 Abbreviated as ZYMV- CpJAZ13, pXT1-ZYMV-eGFP is referred to as ZYMV-eGFP 。 The recombinant overexpression vector ZYMV- CpJAZ13 After the vector was transformed into competent Agrobacterium tumefaciens GV3101 cells, the cells were transferred to LB medium supplemented with 50 μg / mL Kan and 50 μg / mL Rif for expansion. After incubation at 28°C for 16 hours, the pellet was collected by centrifugation at 6000 g for 5 minutes. The pellet was resuspended in a resuspension buffer to prepare the infection medium. After the infection medium was allowed to stand at room temperature for 2 hours, it was injected into zucchini cotyledons using a 1 mL syringe three times. The cells were then incubated at 28°C and observed for fluorescence using a handheld fluorescent flashlight.

[0055] Each liter of the resuspension contains: 10 mmol of MgCl2, 10 mmol of MES, and 100 μmol / L of acetosyringone, and the rest is made up with LB medium.

[0056] SEQ ID NO.1:

[0057] ATGTCGACTTCTTCGGGATTCTCTCAGGTTTCGTCTCTAAAATCGCCGGAGAACCTCGGGTTTCTTCCCGCCGGTGCGATGCTGGTTTCCGGCGAGGATCTGAATCTGGGTTTCTCGGCGAATCTCCGGAGAAGAAACGGGTCGGCGCCGGAACTGTTCCACCGGCCGGAAAAAATGGCGAATCTCAAGTCGTTGGATTTGTTTCCTCAAGAAGCTGGATTTGGTATCTCAAACGACGTCGTTTCAAGCGTCTATAAATCTGCTACGGCGGCGCCTCAAGCTTCCCAAATGACGATCTTTTACGCCGGCCAAGTTTTTGTGTTCAACAATTTCCCGGCGGAAAGAGTCGGCGAAGTTATGTTTCTAGCAGGCCGAGAGAGCTCCAAACTGAACATCCCAGCCGCCACTGTCGCTTCCGGCCAGCCTATCTTGGTCGGAACTCCGGCGGATTCTTTAAGCTCCAGTTCCCCTGTTTCCACTCGGACCCCAACCCCACCACCGCCTCCTCAGCCGCAGTCCGTCACCGGCGCTCTTCCAATGGCTCGTAAAGCTTCGATTCAACGTTTCCTAGAGAAGAGAAGAGACAGGCTAACTCTGAGAGCGCCGTACCAGAACTGCCCGGCGCCGAGCAAACTCGCCGGCGACAACTCCTGGCTGGGTTTGGCCGTGCAGCCGTAG。

[0058] SEQ ID NO.2:

[0059] MSTSSGFSQVSSLKSPENLGFLPAGAMLVSGEDLNLGFSANLRRRNGSAPELFHRPEKMANLKSLDLFPQEAGFGISNDVVSSVYKSATAAPQASQMTIFYAGQVFVFNNFPA ERVGEVMFLAGRESSKLNIPAATVASGQPILVGTPADSLSSSSPVSTRTPTPPPPPQPQSVTGALPMARKASIQRFLEKRRDRLTLRAPYQNCPAPSKLAGDNSWLGLAVQP.

[0060] (4) Determination of reactive oxygen species, antioxidant enzymes and MDA content.

[0061] Determination of relevant indicators, including MDA, H2O2 and O2 - The content levels and CAT, SOD and POD activities were determined using corresponding kits, all of which were purchased from Solarbio.

[0062] (5) Solution preparation.

[0063] Preparation of 1 mg / mL DAB solution: Dissolve 0.1 g DAB in 100 mL distilled water and adjust the pH to 3.8.

[0064] Preparation of 0.5 mg / mL NBT solution: Dissolve 50 mg of NBT in 100 mL of distilled water and adjust the pH to 7.5.

[0065] (6) DAB staining, the method is as follows:

[0066] 1) Transfect the pBI121- CpJAZ13 -eGFP vector tobacco leaves and wild-type tobacco leaves were placed in 1 mg / mL DAB solution and immersed for 8 h.

[0067] 2) Remove the tobacco leaves, place them in 96% ethanol, and boil for 30 minutes to decolorize them.

[0068] 3) Place the decolorized leaves in lactophenol water, make them transparent, and then take photos.

[0069] (7) NBT staining, the method is as follows:

[0070] 1) Transfect the pBI121- CpJAZ13 -eGFP vector tobacco leaves and wild-type tobacco leaves were placed in 0.5 mg / mL NBT solution and immersed for 4 h.

[0071] 2) Remove the tobacco leaves, place them in 96% ethanol, and boil for 30 minutes to decolorize them.

[0072] 3) Place the decolorized transgenic tobacco leaves in lactophenol water, make them transparent, and then take photos.

[0073] 2. Results

[0074] The present invention used Primer 5.0 software to design qRT-PCR primers. The qRT-PCR primers are shown in Table 2. They were used to analyze the genomic DNA of zucchini leaves at 0 h, 6 h, 12 h, and 24 h after treatment with 20% PEG6000. CpJAZ13 gene expression profiles, CpJAZ13 Gene expression profiles Figure 1 qRT-PCR data showed that most CpJAZ The genes all responded to drought stress. CpJAZ13 The expression level of α gradually increased and reached a peak at 12 h after stress. Figure 1 All data were analyzed using the mean ± standard deviation method, and each analysis was repeated at least three times. **p < 0.01.

[0075] For research CpJAZ13 Subcellular localization of pBI121- CpJAZ13 -eGFP and pBI121-eGFP vectors were injected into tobacco epidermal cells respectively and observed under a confocal microscope after incubation for a period of time. CpJAZ13 A prominent green fluorescent signal was observed, which overlapped with the nuclear marker, thus verifying its localization in the cell nucleus. Figure 2 .

[0076] Subsequently, the present invention analyzed the overexpression CpJAZ13 Effects on yeast growth and stress resistance. CpJAZ13 No significant effect on yeast growth, see Figure 3 However, overexpression was observed CpJAZ13 The yeast cells showed obvious resistance in low concentration SD-U medium containing 5% PEG6000, especially at 10 -4 In the case of double infusion.

[0077] ZYMV- CpJAZ13 After the recombinant overexpression vector was transferred into Agrobacterium tumefaciens strain GV3101, zucchini was inoculated using the Agrobacterium-mediated method, and zucchini containing ZYMV-eGFP was used as a control. All zucchini began to show mosaic symptoms on their new leaves 7 days after inoculation. Figure 4 The zucchini plants were placed under a handheld fluorescent light and observed. CpJAZ13 Compared with the zucchini plants inoculated with ZYMV-eGFP, the zucchini plants inoculated with ZYMV-eGFP showed stronger fluorescence throughout the leaves. CpJAZ13 The diseased zucchini plants expressing ZYMV-eGFP and ZYMV-eGFP were successfully infected. Figure 5 qRT-PCR analysis showed that compared with the zucchini plants infected with ZYMV-eGFP, the ZYMV- CpJAZ13 Infected zucchini plants CpJAZ13 The expression level was significantly increased, see Figure 6 . Figure 5 and Figure 6 Among them, OE1, OE2 and OE3 were three strains overexpressing CpJAZ13 zucchini plants; WT1, WT2 and WT3 are three wild-type zucchini plants. Figure 6 All data were analyzed using the mean ± standard deviation method, and each analysis was repeated at least three times. **p < 0.01. The overexpressing plants were subjected to natural drought stress to observe the effect of drought stress on seedling performance. After 4 days of drought, ZYMV-eGFP zucchini plants showed wilting. On the 7th day, ZYMV-eGFP zucchini plants showed obvious wilting, while ZYMV- CpJAZ13 The zucchini plants only showed slight wilting. After watering was resumed in both groups, ZYMV- CpJAZ13 The zucchini plants resumed normal growth, while the ZYMV-eGFP zucchini plants were still wilting after 2 days of watering. Figure 4 Subsequently, the zucchini plants infected with ZYMV-eGFP and ZYMV- CpJAZ13 Infected zucchini plants were subjected to DAB and NBT staining. Figure 7 Drought stress usually causes a lot of oxidative damage in plants. In the present invention, drought stress leads to the O2 - and significant accumulation of H2O2. NBT staining and DAB staining showed that under normal growth conditions, there was no significant difference in leaf color between ZYMV-eGFP and ZYMV-CpJAZ13 lines. However, under drought stress, ZYMV-CpJAZ13 leaves showed weaker blue and brown staining compared with ZYMV-eGFP leaves, indicating that H2O2 and O2 - The accumulation of ZYMV- CpJAZ13 Compared with zucchini plants infected with ZYMV-eGFP, the levels of malondialdehyde, H2O2 and O2 in zucchini plants infected with ZYMV-eGFP were significantly higher than those in zucchini plants infected with ZYMV-eGFP. - The levels of CAT, SOD and POD were higher in drought treatment, while the activities of three antioxidant enzymes, CAT, SOD and POD, were lower. Figure 8 . Figure 8All data were analyzed using the mean ± standard deviation method, and each analysis was repeated at least three times. Different lowercase letters in the bar graph indicate significant differences, p < 0.01. The above results show that overexpression CpJAZ13 The antioxidant activity of zucchini seedlings increased and their resistance to drought stress was enhanced.

[0078] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0079] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.

Claims

1. CpJAZ13 The application of the invention in improving the drought stress resistance of zucchini is characterized in that: described CpJAZ13 The nucleotide sequence is shown in SEQ ID NO.

1.

2. An application of a recombinant overexpression vector, characterized in that: The recombinant overexpression vector is used to construct an overexpression plant to improve the resistance of zucchini to drought stress; the recombinant overexpression vector comprises the CpJAZ13 ; The method for preparing the recombinant overexpression vector comprises the following steps: Total RNA of wild-type zucchini was extracted and reverse transcribed into cDNA; cDNA was used as template and PCR amplified to obtain CpJAZ13 ; After enzyme digestion of the expression vector, seamless cloning was used to CpJAZ13 Connect to the expression vector after enzyme digestion to obtain the recombinant overexpression vector; The expression vector is pXT1-ZYMV-eGFP.

3. The use according to claim 2, characterized in that The method for constructing the overexpression plant comprises the following steps: Transforming the recombinant overexpression vector into Agrobacterium competent cells, expanding the culture, and preparing an infection solution; The zucchini was infected with the infection liquid and then cultured to obtain overexpression plants.

4. The use according to claim 3, characterized in that The Agrobacterium competent cell is GV3101.

5. The use according to claim 3, characterized in that The infected part of the zucchini is the cotyledon.

6. The use according to claim 3, characterized in that The conditions for the expanded culture are culturing at 28° C. for 16 h.

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