A CtVOZ1 protein and gene associated with plant drought resistance and its applications

Abstract

This invention belongs to the field of genetic engineering technology, specifically relating to a CtVOZ1 protein, gene, and applications related to plant drought resistance. This invention provides a CtVOZ1 protein related to plant drought resistance, the amino acid sequence of which is shown in SEQ ID NO.1. The CtVOZ1 protein of this invention can improve plant drought resistance. Examples show that overexpression... CtVOZ1 Genes can increase plant biomass, seed germination rate, chlorophyll content, and maintain photosynthetic efficiency. Compared to wild-type plants, overexpression of these genes... CtVOZ1 Following gene modification, the accumulation of malondialdehyde, superoxide anion, and hydrogen peroxide can be reduced, and the activity of antioxidant enzymes can be significantly increased, thereby improving plant drought resistance. This invention... CtVOZ1 Proteins can yield new germplasm with drought-resistant properties, which is of great significance for breeding drought-resistant crops.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a CtVOZ1 protein and gene related to plant drought resistance and its applications. Background Technology

[0002] In recent years, the global agricultural ecological environment has continued to deteriorate. Drought, as the primary abiotic stress factor, severely inhibits plant seed germination, hinders root and stem growth, and interferes with key physiological processes such as photosynthesis and respiration, leading to a significant decline in crop yields and directly threatening food security. Breeding drought-resistant crops is the core strategy to address this problem, and identifying superior drought-resistant genes is key to molecular breeding.

[0003] As a native plant of desert and arid regions, *Calamus esculenta* has adapted to water-scarce environments over a long period and evolved unique drought-resistant regulatory mechanisms. Its genome contains key drought-resistant genes that have not yet been discovered. However, current research on the screening and functional verification of drought-resistant genes in *Calamus esculenta* is still incomplete, and the application of these genes in crops is even more lacking. Therefore, isolating and identifying drought-resistant genes from *Calamus esculenta* is of great practical significance for enriching plant drought-resistant gene resources and promoting drought-resistant crop breeding. Summary of the Invention

[0004] The purpose of this invention is to provide a CtVOZ1 protein, gene, and application related to plant drought resistance. The CtVOZ1 protein described in this invention positively regulates plant drought resistance and can be used to cultivate drought-resistant plants.

[0005] This invention provides a CtVOZ1 protein related to plant drought resistance, the amino acid sequence of which is shown in SEQ ID NO.1.

[0006] This invention also provides a method related to plant drought resistance. CtVOZ1 Genes, the ones mentioned CtVOZ1 The nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0007] The present invention also provides the CtVOZ1 protein described in the above-described scheme or the [other protein described above]. CtVOZ1 Application of genes in regulating plant drought resistance.

[0008] As a preferred embodiment, the regulation includes: increasing CtVOZ1 protein content or increasing... CtVOZ1 Increased gene expression levels enhance plant drought resistance.

[0009] As a preferred embodiment, the improvement of plant drought resistance includes at least one of the following: (1) increasing germination rate; (2) increasing plant growth; and (3) increasing antioxidant capacity.

[0010] As a preferred embodiment, the enhancement of antioxidant capacity includes at least one of the following: (1) reducing malondialdehyde accumulation; (2) reducing superoxide anion accumulation; (3) reducing hydrogen peroxide accumulation; and (4) enhancing antioxidant enzyme activity.

[0011] As a preferred embodiment, the plant includes *Gnaphalium affine* and / or *Arabidopsis thaliana*.

[0012] The present invention also provides a recombinant expression vector, wherein the recombinant expression vector comprises the components described above. CtVOZ1 Gene.

[0013] The present invention also provides a recombinant bacterium, wherein the recombinant bacterium comprises the recombinant expression vector described in the above scheme.

[0014] The present invention also provides the CtVOZ1 protein described in the above-described scheme, and the aforementioned... CtVOZ1 The application of the gene, the recombinant expression vector, or the recombinant bacteria in the cultivation of new drought-resistant plant germplasm.

[0015] Beneficial Effects: This invention provides a CtVOZ1 protein associated with plant drought resistance, the amino acid sequence of which is shown in SEQ ID NO.1. The CtVOZ1 protein of this invention can improve plant drought resistance. Examples show that overexpression of this protein... CtVOZ1 Genes can increase plant biomass, seed germination rate, chlorophyll content, and maintain photosynthetic efficiency. Compared to wild-type plants, overexpression of these genes... CtVOZ1 Following gene modification, the accumulation of malondialdehyde, superoxide anion, and hydrogen peroxide can be reduced, and the activities of superoxide dismutase, catalase, and peroxidase can be significantly increased, thereby improving plant drought resistance. This invention... CtVOZ1 Proteins can yield new germplasm with drought-resistant properties, which is of great significance for breeding drought-resistant crops. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 ground melon CtVOZ1 Subcellular localization results of genes;

[0018] Figure 2 ground melon CtVOZ1 A graph showing the results of gene transcriptional activation activity;

[0019] Figure 3 ground melon CtVOZ1Results of functional validation of gene-overexpressing yeast; where A and B represent the growth and concentration (OD) of overexpressing yeast and empty vector yeast on culture medium under normal conditions. 600 Figures C and D show the growth and concentration of overexpressed yeast and empty vector yeast on the culture medium under drought stress. 600 Figure; specifically, A and C correspond to results on solid culture medium, and B and D correspond to results on liquid culture medium;

[0020] Figure 4 ground melon CtVOZ1 Figure 1 shows the results of constructing the Arabidopsis thaliana vector for gene overexpression; where A is a schematic diagram of vector construction; B is a gel electrophoresis result, where 1458bp is the gene size and 1607bp is the PCR length using the gene forward primer and the vector reverse primer.

[0021] Figure 5 ground melon CtVOZ1 Figure showing the results of gene overexpression analysis in Arabidopsis thaliana plants;

[0022] Figure 6 ground melon CtVOZ1 Germination of Arabidopsis thaliana seeds with overexpressed genes under drought stress; where A is the germination of the control group; B is the germination of the drought-stressed group; C is the germination rate of the control group; and D is the germination rate of the drought-stressed group.

[0023] Figure 7 ground melon CtVOZ1 Phenotypic analysis results of Arabidopsis thaliana plants with overexpressing genes under stress;

[0024] Figure 8 ground melon CtVOZ1 Figure showing the results of fresh weight and chlorophyll content analysis of Arabidopsis thaliana plants with overexpressing genes under stress;

[0025] Figure 9 ground melon CtVOZ1 Figure showing the results of physiological index analysis of Arabidopsis thaliana plants with overexpressing genes under stress;

[0026] Figure 10 ground melon CtVOZ1 Identification results of gene-silenced plants; where A represents the PCR identification results of the silenced fragment; and B represents the analysis results of the expression level of the silenced plants.

[0027] Figure 11 ground melon CtVOZ1 Phenotypic analysis results of gene-silenced plants under stress;

[0028] Figure 12 ground melon CtVOZ1 Analysis results of fresh weight and chlorophyll content of gene-silenced plants under stress;

[0029] Figure 13 ground melon CtVOZ1 Analysis results of relevant physiological indicators of gene-silenced plants under stress;

[0030] In the picture This indicates that the data shows a significant difference. p <0.01. Detailed Implementation

[0031] This invention provides a CtVOZ1 protein associated with plant drought resistance, the amino acid sequence of which is shown in SEQ ID NO.1: MGKGSKSGACKSASHQLFKDRAKNRVDDLQGMFSDLQSARKESRTIDVAVLEEQVHQMLREWKAELNEPSPASSLQGGSLGTFSSDIYRLLQLCEEEDDATSALTPPKSEPGTQKVDSGAVFQEGFNSCHVPQEQSFQLLDQCKTSPSGANTMGINNMGIASHIDYHPFDLHQEFDHQYFTGFDGTGICGEDALTQISNFSQNYCPPPAAFLGPKCALWDCPRPAQRSEWCQTSQDYCS The CtVOZ1 protein described in this invention is located in the cell nucleus and can effectively reduce the accumulation of ROS under drought stress by improving the plant's antioxidant capacity and maintaining photosynthetic efficiency. This reduces oxidative damage and enhances the plant's drought tolerance, which is of great significance for cultivating new drought-resistant germplasm.

[0032] This invention also provides a method related to plant drought resistance. CtVOZ1 Genes, the ones mentioned CtVOZ1

[0033] The present invention also provides the CtVOZ1 protein described in the above-described scheme or the [other protein described above]. CtVOZ1 Application of genes in regulating plant drought resistance.

[0034] As one implementation method, the regulation includes: increasing CtVOZ1 protein content or increasing... CtVOZ1 Increased gene expression levels enhance plant drought resistance.

[0035] As one implementation method, the improvement of plant drought resistance includes at least one of the following: (1) increasing germination rate; (2) increasing plant growth; and (3) increasing antioxidant capacity.

[0036] As one implementation, the enhancement of antioxidant capacity includes at least one of the following: (1) reducing malondialdehyde accumulation; (2) reducing superoxide anion accumulation; (3) reducing hydrogen peroxide accumulation; and (4) increasing antioxidant enzyme activity. As one implementation, the antioxidant enzyme includes at least one of superoxide dismutase, catalase, and peroxidase. Example results show that overexpression... CtVOZ1 Compared to the wild type, the strains showed significantly lower accumulations of malondialdehyde, superoxide anion, and hydrogen peroxide, and higher activities of superoxide dismutase, catalase, and peroxidase. CtVOZ1 By enhancing antioxidant capacity and maintaining photosynthetic efficiency, the accumulation of ROS under drought stress is effectively reduced, thereby mitigating oxidative damage and improving the drought resistance of plants.

[0037] In one embodiment, the plant includes *Gnaphalium affine* and / or *Arabidopsis thaliana*.

[0038] The present invention also provides a recombinant expression vector, wherein the recombinant expression vector comprises the components described above. CtVOZ1 Gene. As one implementation, the base vector of the recombinant expression vector includes pYES2.

[0039] The present invention also provides a recombinant bacterium, characterized in that the recombinant bacterium comprises the recombinant expression vector described in the above-described scheme. As one embodiment, the base bacterium of the recombinant bacterium includes Agrobacterium. In a specific embodiment of the present invention, the base bacterium of the recombinant bacterium is Agrobacterium GV3103.

[0040] The present invention also provides the CtVOZ1 protein described in the above-described scheme, and the aforementioned... CtVOZ1 The application of the gene, the recombinant expression vector, or the recombinant bacteria in the cultivation of new drought-resistant plant germplasm.

[0041] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0042] Example 1 CtVOZ1 Subcellular localization and transcriptional activation

[0043] (1) CtVOZ1 Subcellular localization

[0044] Build CtVOZ1 The fusion expression vector with green fluorescent protein (GFP) is processed as follows:

[0045] ① Using *Citrus aurantiacus* genomic DNA as a template, PCR amplification was performed using primers CtVOZ1-eGFP-F (SEQ ID NO.5) and CtVOZ1-eGFP-R (SEQ ID NO.6) listed in Table 1 to obtain samples containing... CtVOZ1 The target segment of a gene.

[0046] Table 1 Primer sequence information

[0047]

[0048] ② The PCAMBIA1300-35S-eGFP vector (Beijing Cooler Master Technology Co., Ltd., product number: VT119, denoted as 35S:eGFP) was double-digested with restriction endonucleases KpnI and XbaI, and the linearized PCAMBIA1300-Egfp vector was recovered.

[0049] ③The ingredients contained in step ① CtVOZ1 The target fragment of the gene and step ② linearize the PCAMBIA1300-35S-eGFP vector for homologous recombination to obtain the recombinant vector, denoted as 35S:CtVOZ1-eGFP.

[0050] ④ The recombinant vector was transferred into Agrobacterium GV3103 to obtain recombinant Agrobacterium bacterial suspension.

[0051] ⑤ The OD of the recombinant Agrobacterium bacterial suspension was determined by resuspension. 600 The value was adjusted to 0.8 to obtain the inoculum; the resuspension consisted of the following components at the following concentrations: MgCl2·6H2O 2.033 g / L, 2-(N-morpholine)ethanesulfonic acid (MES) 2.132 g / L and acetylsyl syringone 200 µM.

[0052] ⑥ Mark the leaves with numbers, and use a 1mL syringe to inject the infection solution and the nuclear localization marker mCherry (concentration of OD) from the back of the leaf using pressure. 600=0.8) was injected into tobacco leaves at a volume ratio of 1:1, then the leaves were sprayed with water, covered with a plastic bag, and placed in the dark overnight. On the second day, the plastic bag was opened, and the leaves were observed under a laser confocal microscope; an empty vector (35S:eGFP) was set up as a control group. The results are shown below. Figure 1 From left to right: green fluorescent protein, cell nuclear localization signal, merged image and bright field image, scale bar is 20 μm.

[0053] The results showed that the fluorescence signal of the CtVOZ1-GFP fusion protein appeared only in the cell membrane, while the fluorescence signal of the empty vector control appeared in both the cytoplasm and the nucleus, indicating that the CtVOZ1 protein is localized in the cell membrane.

[0054] (2) To further verify the self-activation characteristics of CtVOZ1, a yeast self-activation experiment was conducted, with the following steps:

[0055] ① Construct the CtVOZ1-pGBKT7 recombinant plasmid. The construction method is similar to that of the recombinant vector 35S:CtVOZ1-eGFP in step (1). The difference is that the primers used to amplify the target fragment are CtVOZ1-BDF (SEQ ID NO.7) and CtVOZ1-BDR (SEQ ID NO.8) in Table 1. The PCAMBIA1300-eGFP vector is replaced with the pGBKT7 vector (purchased from Beijing Cooler Master Technology Co., Ltd., product number: VT006). The restriction endonucleases are EcoRI and BamHI.

[0056] ② The CtVOZ1-pGBKT7 recombinant plasmid and the empty vector pGBKT7 (negative control) were transformed into yeast Y2Hgold, respectively. After transformation, the yeast was cultured on a medium lacking tryptophan (denoted as SD-Trp) and a medium lacking tryptophan, histidine, and adenine (denoted as SD-Trp-Ade-His). The SD-Trp was purchased from Beijing Cooler Technology Co., Ltd., product number: PM2252; the SD-Trp-Ade-His was purchased from Beijing Cooler Technology Co., Ltd., product number: PM2302.

[0057] The results are as follows Figure 2 As shown in the figure. The results showed that both the CtVOZ1-pGBKT7 recombinant plasmid and the empty vector could grow normally on SD-Trp medium; and neither could grow on SD-Trp-Ade-His medium, indicating that CtVOZ1 does not exhibit self-activation.

[0058] Example 2 Overexpression CtVOZ1 It enhanced the yeast's resistance to drought stress.

[0059] In containing CtVOZ1 Overexpression vector (pYES2-) CtVOZ1 In yeast, analysis was performed. CtVOZ1 The effects of proteins on yeast growth and stress resistance were investigated through the following steps:

[0060] ①Construction CtVOZ1 The overexpression vector was constructed using the same method as the recombinant vector 35S in step (1) of Example 1. CtVOZ1 Similar to -eGFP, the difference lies in the primers used to amplify the target fragment, which are pYES2- from Table 1. CtVOZ1 -F (SEQ ID NO.3) and pYES2- CtVOZ1 -R (SEQ ID NO.4), replacing the PCAMBIA1300-eGFP vector with the pYES2 vector (purchased from Beijing Coollife Technology Co., Ltd., product number: VT064), with restriction endonucleases BamHI and EcoRI.

[0061] ② Take 100µL of INVSC1 competent cells thawed on ice (purchased from Beijing Coolplay Technology Co., Ltd., product number: CC303), and add the pre-cooled target plasmid (pYES2-) sequentially. CtVOZ1 Add 2~5µg of pYES2), 10µL of carrier DNA, and 500µL of PEG / LiAc. Mix well by pipetting several times, then incubate at 30℃ for 30 min and at 42℃ for 15 min.

[0062] ③ Centrifuge at 10,000 rpm for 30 seconds, discard the supernatant, resuspend in 400 µL of ddH2O, centrifuge for 30 seconds, and discard the supernatant.

[0063] ④ Resuspend the culture in 50 µL of ddH2O, plate it onto an SD / -Ura plate, screen for positive clones, and then inoculate it into 15 mL of SD / -Ura Broth yeast auxotrophic liquid medium. Incubate overnight at 30°C with a shaker. The SD / -Ura plates were purchased from Beijing Cooler Master Technology Co., Ltd., product number: PM2272; the SD / -Ura Broth yeast auxotrophic liquid medium was purchased from Beijing Cooler Master Technology Co., Ltd., product number: PM2271.

[0064] ⑤ Centrifuge at 1500g for 5 minutes at 4℃ and remove the supernatant.

[0065] ⑥ Resuspend the cell pellet in 50 mL of SG / -Ura liquid medium and incubate in a shaker at 30 °C.

[0066] ⑦OD 600 After adjusting to 1 and serially diluting 10-fold, the growth of SD / -Ura Broth yeast was evaluated on growth-deficient solid and liquid media containing different concentrations of drought stress (0 mM, designated as control and 60 mM PEG3350). The results are as follows: Figure 3 As shown in Table 2, Figure 3 In the diagram, A and B represent the OD values ​​of overexpressed yeast and empty vector yeast on the culture medium under normal conditions. 600 Figures C and D show the growth and concentration of overexpressed yeast and empty vector yeast on the culture medium under drought stress. 600 In the figure, A and C correspond to the results of solid culture medium, and B and D correspond to the results of liquid culture medium.

[0067] Table 2. Functional validation data of yeast overexpressing CtVOZ1 gene from *Cucumis melo*.

[0068]

[0069] The results showed that CtVOZ1 Overexpression had no significant effect on yeast growth. Figure 3 (A) CtVOZ1 Yeast cells overexpressing PEG3350 exhibited significant resistance in medium containing 60 mM PEG3350, especially at a 100-fold dilution. Figure 3 (C). Furthermore, under liquid culture conditions (SD / -Ura Broth yeast auxotrophic liquid medium), the transformed... CtVOZ1 Survival tests on the yeast strains revealed that, under normal conditions without 60 mM PEG3350, the growth trends of the two yeast strains were essentially the same. Figure 3 (B) Under drought stress, CtVOZ1 The survival rate of the overexpressing yeast strain was consistently higher than that of the control group. Specifically, under stress at a level of 60 mM PEG3350, after 9 hours of stress treatment, CtVOZ1 The survival rate of the overexpressing yeast strain was significantly higher than that of the empty vector control strain. Figure 3 (D). These results indicate that CtVOZ1 The protein enhances the yeast's ability to resist drought stress.

[0070] Example 3 CtVOZ1 Genetic heterologous transformation of Arabidopsis thaliana and screening of positive plants

[0071] Constructed using the method of Example 1 CtVOZ1 The plant expression vector 35S:CtVOZ1-eGFP, and its construction diagram are shown below. Figure 4 As shown in Figure A, Arabidopsis thaliana (Columbia type) was transformed using an inflorescence immersion method mediated by Agrobacterium GV3103. T0 generation transgenic plants were screened on 1 / 2 MS medium containing 50 mg / L HYG (hygromycin). T1 and T2 generations were screened consecutively using the same method to obtain homozygous transgenic lines.

[0072] To verify the transgenic plants, homozygous samples were extracted in this embodiment. CtVOZ1 Genomic DNA from transgenic Arabidopsis and wild-type Arabidopsis was used. CtVOZ1 PCR amplification was performed using specific primers (CtVOZ1-eGFP-F (SEQ ID NO. 5) and p1300-R (SEQ ID NO. 11) listed in Table 1). The PCR reaction system was as follows: 25 μL of 2×TransStart® FastPfu FlyReaction Mix, 1 μL of TransStart® FastPfu Fly DNA Polymerase, 1 μL each of forward and reverse primers, 1 μL of cDNA, and 21 μL of ddH2O. The reaction program was: 98℃ for 1 min; 98℃ for 10 s, 60℃ for 5 s, 72℃ for 10 s, for 30 cycles; 72℃ for 1 min; and stored at 4℃. The results are as follows. Figure 4 As shown in B, where +: with CtVOZ1 The amplification product of the plant expression vector template; OE1-6: CtVOZ1 Transgenic lines; WT wild-type Arabidopsis thaliana. All were identified as positive plants.

[0073] Simultaneously, total RNA was extracted from Arabidopsis transgenic homozygous lines that were positive for HYG screening and molecular identification. Using primers CtVOZ1-qPCR-F (SEQ ID NO.9) and CtVOZ1-qPCR-R (SEQ ID NO.10) listed in Table 1, qRT-PCR was performed to detect RNA. CtVOZ1 For gene expression levels, the primers for the internal reference genes were Atactin-F (SEQ ID NO.14) and Atactin-R (SEQ ID NO.15) listed in Table 1. The qRT-PCR reaction system was as follows: 12.5 μL of TB Green Premix Ex Taq II FastqPCR (2X), 1 μL each of forward and reverse primers, 1 μL of cDNA, and 9.5 μL of ddH2O. The reaction program was as follows: 25℃ for 10 min; 95℃ for 30 s; 95℃ for 5 s, 60℃ for 10 s, for 40 cycles; 95℃ for 15 s; 60℃ for 1 min; 95℃.

[0074] See results Figure 5 As shown in Table 3, the results indicate that the overexpressing plants were significantly different from the WT plants. p <0.01). This indicates that the overexpressing plants were successfully heterologously transformed. CtVOZ1 The positive plants for the gene provided reliable experimental material for subsequent functional analysis.

[0075] Table 3. Expression levels of CtVOZ1 gene overexpression in Arabidopsis thaliana plants.

[0076]

[0077] Example 4 CtVOZ1 Overexpression enhances drought tolerance in Arabidopsis thaliana

[0078] Mature T3 generation seeds and wild-type (WT) Arabidopsis seeds were collected, air-dried, and vernalized at 4℃ for 2 days (in a 4℃ incubator). After vernalization, the seeds were treated with 70% ethanol for 10 min consecutively, followed by anhydrous ethanol for 20 min consecutively, to sterilize the seed surface. The sterilized Arabidopsis seeds were sown on 1 / 2 MS solid medium (10cm × 10cm square plates) and randomly divided into two groups. One group was subjected to drought stress with 30mM PEG3350 (drought stress group); the other group was subjected to drought stress without PEG3350 (control group). Both groups of seeds were placed in a growth chamber at 24℃, 16 h light, 8 h dark, and 70% relative humidity for germination experiments, with three replicates per group. Germination rate was recorded every 24 h. The results are shown below. Figure 6 As shown in Table 4.

[0079] Transgenic Arabidopsis thaliana seedlings with consistent growth and WT wild-type seedlings were randomly divided into two groups: a control group and a drought stress group. The drought stress group was deprived of water for 14 days to simulate drought stress and observe its growth under drought conditions. Results are shown below. Figure 7 As shown.

[0080] Table 4 Germination of Arabidopsis seeds overexpressing the CtVOZ1 gene

[0081]

[0082] The results showed that under normal growth conditions, there were no significant differences in growth status and phenotype between wild-type and overexpression lines. However, after drought stress treatment, the growth of both wild-type and overexpression lines was significantly inhibited, manifested as stunted growth and yellowing of leaves due to dehydration, although the overexpression lines experienced less inhibition. Under drought stress, although 30 mM PEG3350 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. These results indicate that... CtVOZ1 Overexpression of this substance significantly enhanced the plant's resistance to drought stress. This indicates that... CtVOZ1 It plays an important role in the response to drought stress.

[0083] Example 5 CtVOZ1 Enhanced physiological indicators and antioxidant capacity of transgenic Arabidopsis thaliana

[0084] In order to conduct a comprehensive assessment CtVOZ1 The role of Arabidopsis thaliana in drought tolerance: This example focuses on plants grown in nutrient soil. CtVOZ1 The transgenic lines were subjected to drought stress treatment, as follows:

[0085] Mature T3 generation seeds and wild-type (WT) Arabidopsis seeds were collected, air-dried, and vernalized at 4℃. Before sowing onto 1 / 2 MS solid medium, the seeds were surface-sterilized by treatment with 70% ethanol for 10 min followed by anhydrous ethanol for 20 min. The sterilized seeds were sown in 1 / 2 MS medium and transferred to a growth chamber at 24℃, with 16 h of light, 8 h of darkness, and 70% relative humidity. At four true leaves, the seeds were transplanted into a 1:3 (v / v) mixture of potting soil and vermiculite. Fourteen days after transplanting, the seeds were subjected to drought stress and watering was stopped for 14 days. After 14 days of cultivation, the seedlings were observed, fresh weight was recorded, and chlorophyll content was measured. The results are shown below. Figure 8 As shown in Table 5.

[0086] Drought stress typically leads to excessive accumulation of reactive oxygen species (ROS) in plants, causing severe oxidative damage. This example also uses a kit from Beijing Solarbio Science & Technology Co., Ltd. to detect the content of superoxide anion (H2O2) and hydrogen peroxide (O2) in Arabidopsis leaves. 2- The content of malondialdehyde (MDA), catalase (CAT) activity, superoxide dismutase (SOD) activity, and peroxidase (POD) activity were measured, and the results are as follows: Figure 9 As shown in Table 6.

[0087] Table 5. Analysis of fresh weight and chlorophyll content of Arabidopsis thaliana plants overexpressing the CtVOZ1 gene under stress.

[0088]

[0089] Table 6. Analysis of relevant physiological indicators of Arabidopsis plants overexpressing the CtVOZ1 gene under stress.

[0090]

[0091] Under normal conditions, there were no significant changes in leaf fresh weight and chlorophyll content between wild-type and overexpression lines, while under stress, the fresh weight and chlorophyll content of the overexpression lines were significantly higher than those of the wild-type lines. Figure 8 Drought stress significantly increased the levels of malondialdehyde (MDA), superoxide anion, and hydrogen peroxide in Arabidopsis leaves. Compared with wild-type plants, the overexpression lines showed significantly lower accumulations of MDA, superoxide anion, and hydrogen peroxide. Figure 9Drought stress significantly increased the activities of superoxide dismutase, catalase, and peroxidase in both wild-type and overexpression lines. The activities of these antioxidant enzymes were significantly higher in the overexpression lines compared to the wild-type. Figure 9 ).

[0092] These results indicate that CtVOZ1 By enhancing antioxidant capacity and maintaining photosynthetic efficiency, the accumulation of ROS under drought stress was effectively reduced, thereby mitigating oxidative damage and improving the drought resistance of plants.

[0093] Example 6 CtVOZ1 Silent carrier construction

[0094] In containing CtVOZ1 Silencing vector (pTRV2-) CtVOZ1 In the ground-top melons, analysis was performed. CtVOZ1 The effects of protein on the growth and stress resistance of ground-shoot cucumbers were investigated through the following steps:

[0095] Build CtVOZ1 The method for constructing the silencing vector is the same as that for the recombinant vector 35S in Example 1: CtVOZ1 Similar to -eGFP, the difference lies in the primers used to amplify the target fragment, which are pTRV2- from Table 1. CtVOZ1 -F (SEQ ID NO.12) and pTRV2- CtVOZ1 -R (SEQ ID NO.13) replaces the PCAMBIA1300-eGFP vector with the pTRV2 vector, and the restriction endonucleases are XbaⅠ and KpnⅠ.

[0096] TRV vector systems typically contain two plasmids: pTRV1 (helper vector, providing replication and movement proteins) and pTRV2 (vector backbone, used for inserting the target gene fragment). The target gene fragment needs to be cloned into pTRV2, working together with pTRV1 to achieve silencing. Fragment selection: A specific 300bp fragment of the target gene is selected. The ligation product is transformed into E. coli, positive clones are selected by antibiotic resistance plate screening, and after sequencing verification, the recombinant pTRV2 plasmid is extracted (pTRV1 plasmid is prepared simultaneously).

[0097] The TRV2 plasmid was double-digested with restriction endonucleases KpnI and Xba1. The vector was ligated using a homologous recombination kit and transformed into competent *E. coli* cells. After antibiotic selection, positive single colonies were picked and detected by PCR using vector primers. The TRV2-Ct vector successfully ligated the insert fragment... VOZ1 The product obtained by PCR amplification of the recombinant plasmid is 300 bp in length (the insert fragment is 300 bp in length). Figure 10 In A, where +: TRV2-Ct VOZ1Recombinant plasmid; pTRV:CtVOZ1: silent plant; WT: wild-type ground squash), the inserted fragment sequence is shown in SEQ ID NO.16: 5'-TCGAAGAGCATTTGAAAGTGGAAACAGAAAGCAAAGGTCATTGCCAGATTATAATGGACGGGGCTGGCATGAATCTAGGAAACAAGTGATGAATGAGTTTGGAGGATTAAAAAGATCATATTATATGGATCCACAACCAATGAAGACTTATGAGTGGCATCTCTATGAATATGAGATCAACAAATATGATGCTTGTGCATTGTACAGATTGGAAATAAAGTTTGTTGACGGGAAAAAGAGTCCAAAAGGTAAAGTAGCTAATGATTCAGTTGCTGATCTGCAGAAGCAAATGAAAAAGCT-3'.

[0098] Will carry TRV1 and TRV2-Ct VOZ1 Agrobacterium GV3101 was cultured overnight at 28°C with shaking at 180 rpm in LB medium containing kanamycin (50 μg / mL) and rifampin (25 μg / mL). The cells were collected by centrifugation, resuspended in an invasion dye solution (containing 10 mmol / L MgCl2, 10 mmol / L LMES, and 200 μmol / L acetylsyleugenone), and the OD600 was adjusted to approximately 1.0. The cells were then incubated in the dark for 4–6 hours in preparation for injection infection.

[0099] Take equal volumes of pTRV1 Agrobacterium resuspension and recombinant viral vector pTRV2-Ct VOZ1Agrobacterium resuspension was thoroughly mixed and used for injection infection. Ground elm seedlings cultured to the cotyledon stage in a light incubator were used as the infection material. A disposable 1mL needleless syringe was used to inject the bacterial solution into the apical meristem of the seedlings. Before injection infection, slight wounds could be created before injecting the bacterial solution into the apical meristem. Infected plants were then cultured in a light incubator at 22℃, 50% humidity, and a photoperiod of L:D-16h:8h. The expression level of CtVOZ1 was detected by RT-qPCR. The primers used are shown in Table 1: CtVOZ1-qPCR-F (SEQ ID NO. 9) and CtVOZ1-qPCR-R (SEQ ID NO. 10). The qRT-PCR reaction system was as follows: 12.5 μL of TB Green PremixEx Taq II Fast qPCR (2X), 1 μL of each of the forward and reverse primers, 1 μL of cDNA, and 9.5 μL of ddH2O. The reaction program was as follows: 25℃ for 10 min, 95℃ for 30 s, [95℃ for 5 s, 60℃ for 10 s] for 40 cycles, 95℃ for 15 s, 60℃ for 1 min, and 95℃.

[0100] Test results as follows Figure 10 As shown in Table B and Table 7, the results indicate that the line with the silenced CtVOZ1 gene was successfully constructed.

[0101] Table 7. Analysis of CtVOZ1 gene expression levels in *Cucumis melo* var. *mongolica*.

[0102]

[0103] Example 7: Silence CtVOZ1 It reduced the resistance of ground-shoot melons to drought stress.

[0104] Operational investigation based on Example 5 CtVOZ1 The phenotype, physiological indicators, and antioxidant capacity of *Melon simonii* after gene silencing were analyzed, and the results are as follows: Figures 11-13 As shown in Tables 8 and 9.

[0105] Table 8. Analysis of fresh weight and chlorophyll content of *Cucumis melo* CtVOZ1 gene-silenced plants under stress.

[0106]

[0107] Table 9. Analysis of relevant physiological indicators of CtVOZ1 gene-silenced plants of *Cucumis melo* under stress.

[0108]

[0109] TRV-mediated ground cucumber CtVOZ1In gene silencing experiments, under normal control conditions, there were no significant differences in growth phenotype between wild-type (WT), empty vector TRV-infected plants, and pTRV2-CtVOZ1 plants; however, after drought stress treatment, the growth of pTRV2-CtVOZ1 plants was more significantly inhibited, exhibiting more severe wilting symptoms. Figure 11 Physiological analysis further showed that the fresh weight and chlorophyll content of pTRV2-CtVOZ1 plants under drought stress were significantly lower than those of WT and TRV plants. Figure 12 ), malondialdehyde (MDA), superoxide anion (O2) 2- Oxidative damage indicators such as hydrogen peroxide (H2O2) were significantly elevated, while the antioxidant enzyme activities of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) were significantly reduced. Figure 13 ).

[0110] In conclusion, CtVOZ1 This gene plays a crucial role in regulating drought stress tolerance in *Calamus stenoptera*. Its silencing affects the activity of antioxidant enzyme systems and the degree of oxidative damage, leading to decreased drought stress tolerance in plants. Overexpression within the plant... CtVOZ1 Genes can improve a plant's drought resistance.

[0111] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A CtVOZ1 protein associated with plant drought resistance, the amino acid sequence of which is shown in SEQ ID NO.

1.

2. A CtVOZ1 gene associated with plant drought resistance, the nucleotide sequence of which is shown in SEQ ID NO.

2.

3. The application of the CtVOZ1 protein of claim 1 or the CtVOZ1 gene of claim 2 in regulating plant drought resistance; The regulation is to improve plant drought resistance by increasing the content of CtVOZ1 protein or increasing the expression of CtVOZ1 gene; the plant is Cucurbita spp. and / or Arabidopsis thaliana.

4. The application according to claim 3, characterized in that, The improvement of plant drought resistance includes at least one of the following: (1) increasing germination rate; (2) increasing plant growth; and (3) improving antioxidant capacity.

5. The application according to claim 4, characterized in that, The improvement of antioxidant capacity includes at least one of the following: (1) reducing malondialdehyde accumulation; (2) reducing superoxide anion accumulation; (3) reducing hydrogen peroxide accumulation; and (4) increasing antioxidant enzyme activity.

6. A recombinant expression vector, characterized in that, The recombinant expression vector contains the CtVOZ1 gene as described in claim 2.

7. A recombinant bacterium, characterized in that, The recombinant bacteria comprises the recombinant expression vector of claim 6.

8. The application of the CtVOZ1 protein of claim 1, the CtVOZ1 gene of claim 2, the recombinant expression vector of claim 6, or the recombinant bacteria of claim 7 in the cultivation of new drought-resistant plant germplasm; wherein the plant is Cucurbita spp. and / or Arabidopsis thaliana.

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