Application of cotton GhNaKR3 gene in improving drought resistance of plants

Abstract

The application discloses cotton GhNaKR3 Gene in improving plant drought resistance belongs to the technical field of plant genetic engineering. GhNaKR3 The CDS sequence of the gene is shown as SEQ ID NO:1, and the application is realized by overexpressing the GhNaKR3 gene in Arabidopsis. GhNaKR A pCAMBIA1302::GhNaKR3 recombination expression vector is constructed, which is transformed into Agrobacterium tumefaciens GV3101, and the Arabidopsis is transformed by the flower inflorescence dipping method. GhNaKR3 The overexpression of the GhNaKR3 gene improves the drought resistance of the Arabidopsis under mannitol simulated drought and natural drought conditions. The application further discloses a method for improving the drought resistance of Arabidopsis, a recombination expression vector and recombination agrobacterium.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to cotton. GhNaKR3 The application of genes in improving plant drought resistance. Background Technology

[0002] Drought stress is one of the important abiotic stresses affecting plant growth and development and crop yield. When plants are subjected to drought stress, their cellular water balance, photosynthesis, metabolism, and antioxidant defense systems are all affected. In severe cases, it can lead to inhibited plant growth, wilting leaves, reduced chlorophyll content, and even plant death.

[0003] Cotton is an important economic crop. Drought stress adversely affects the vegetative growth, reproductive growth, photosynthesis, and fiber formation of cotton. Therefore, identifying the functional genes involved in the drought stress response of cotton and clarifying their roles in the regulation of plant drought resistance is of great significance for the study of plant drought resistance mechanisms and the creation of drought-resistant materials.

[0004] NaKR proteins are a class of plant proteins containing heavy metal-associated domains. Arabidopsis NaKR3 has been reported to participate in salt stress responses, but NaKR family members from different plant sources may exhibit functional differentiation. (Cotton...) GhNaKR3 The function of genes in drought stress response and whether they can be used to improve plant drought resistance still need further investigation.

[0005] This invention analyzes GhNaKR3 The expression patterns of genes under drought stress were investigated, and their role in drought resistance in cotton was verified by using virus-induced gene silencing technology. This was further validated by overexpression in Arabidopsis thaliana. GhNaKR3 The gene underwent positive function verification, which clarified that... GhNaKR3 The gene enhances the drought resistance of plants, and its overexpression in Arabidopsis thaliana has been demonstrated. GhNaKR3 Genes can enhance the drought resistance of Arabidopsis thaliana. Summary of the Invention

[0006] The purpose of this invention is to provide cotton GhNaKR3 The application of genes in improving plant drought resistance, clearly GhNaKR3 The gene's drought resistance regulatory function, and provides a method to overexpress it in Arabidopsis. GhNaKR3 Methods to enhance drought resistance in Arabidopsis thaliana through gene therapy.

[0007] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides cotton. GhNaKR3 The application of genes in improving plant drought resistance, the aforementioned GhNaKR3The CDS sequence of the gene is shown in SEQ ID NO: 1; the application is achieved by overexpressing the gene in plants. GhNaKR3 Genetic implementation; the plant in question is Arabidopsis thaliana.

[0008] Furthermore, the overexpression is achieved using the pCAMBIA1302::GhNaKR3 recombinant expression vector; the recombinant expression vector is obtained by transcribing the vector shown in SEQ ID NO: 1. GhNaKR3 The coding sequence in the gene CDS sequence, except for the 3′ stop codon TGA, was ligated into the pCAMBIA1302 vector and then ligated in the same frame with the mGFP5 coding sequence in the vector.

[0009] Further, the pCAMBIA1302::GhNaKR3 recombinant expression vector was transformed into Agrobacterium tumefaciens GV3101.

[0010] Furthermore, Agrobacterium tumefaciens GV3101 carrying the pCAMBIA1302::GhNaKR3 recombinant expression vector was used to transform Arabidopsis thaliana via inflorescence immersion.

[0011] Furthermore, the Arabidopsis thaliana is the Colombian ecotype.

[0012] Secondly, the present invention provides a method for improving the drought resistance of Arabidopsis thaliana, comprising the following steps: (1) The sample shown in SEQ ID NO: 1 GhNaKR3 The coding sequence in the gene CDS sequence, except for the 3′ stop codon TGA, was ligated into the pCAMBIA1302 vector and co-linked with the mGFP5 coding sequence in the vector to obtain the pCAMBIA1302::GhNaKR3 recombinant expression vector. (2) The pCAMBIA1302::GhNaKR3 recombinant expression vector was transformed into Agrobacterium tumefaciens GV3101; (3) Agrobacterium tumefaciens GV3101 carrying the pCAMBIA1302::GhNaKR3 recombinant expression vector was used to transform Arabidopsis thaliana by inflorescence infiltration method; (4) The Arabidopsis seeds obtained after transformation were screened for hygromycin resistance, and the screened plants were identified by PCR. GhNaKR3 Gene expression level detection to obtain overexpression GhNaKR3 Arabidopsis thaliana plants with genetic material.

[0013] Further, in step (3), the OD of the resuspension of Agrobacterium tumefaciens GV3101 is... 600 The value ranges from 0.8 to 1.2.

[0014] Furthermore, the resuspension contains 0.02% Silwet-77 by volume.

[0015] Furthermore, the Arabidopsis inflorescence was immersed in the resuspension for 2–3 seconds, with humidity maintained above 90%, and cultured in the dark at 25°C for 24 hours.

[0016] Furthermore, the inflorescence was dipped at intervals of 7 days, for a total of 3 dips.

[0017] Furthermore, the hygromycin resistance screening was performed using 1 / 2 MS medium containing 35 mg / L hygromycin B.

[0018] Thirdly, the present invention provides a recombinant expression vector, wherein the recombinant expression vector is the vector shown in SEQ ID NO: 1. GhNaKR3 The coding sequence in the gene CDS sequence, except for the 3′ stop codon TGA, was ligated into the pCAMBIA1302 vector and then ligated in the same frame with the mGFP5 coding sequence in the vector to obtain the pCAMBIA1302::GhNaKR3 recombinant expression vector.

[0019] Fourthly, the present invention provides a recombinant Agrobacterium, wherein the recombinant Agrobacterium is Agrobacterium tumefaciens GV3101 carrying the above-mentioned pCAMBIA1302::GhNaKR3 recombinant expression vector.

[0020] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention silences the VIGS in cotton through a TRV-mediated system. GhNaKR3 Genetic analysis revealed that gene silencing in cotton plants exacerbated wilting under drought stress, increased malondialdehyde (MDA) levels, and decreased catalase activity and chlorophyll content, indicating... GhNaKR3 The gene is a positive regulator of drought resistance in cotton.

[0021] (2) In this invention, a recombinant expression vector pCAMBIA1302::GhNaKR3 was constructed and introduced into Arabidopsis thaliana. Arabidopsis thaliana overexpressing the GhNaKR3 gene showed strong drought resistance under mannitol-simulated drought and natural drought conditions, proving that overexpression in Arabidopsis thaliana is effective. GhNaKR3 Genes can enhance the drought resistance of Arabidopsis thaliana.

[0022] (3) This invention provides the pCAMBIA1302::GhNaKR3 recombinant expression vector and Agrobacterium tumefaciens GV3101 containing the recombinant expression vector, which can be used for... GhNaKR3 This provides a technological foundation for research on drought-resistant gene functions and the creation of drought-resistant plant materials. Attached Figure Description

[0023] Figure 1The image shows the predicted three-dimensional structure of the GhNaKR3 protein.

[0024] Figure 2 Figure 1 shows the phylogenetic analysis and amino acid sequence alignment results of GhNaKR3 protein and its homologs. In Figure 2, A is the phylogenetic tree of GhNaKR3 protein and its homologs, and B is the amino acid sequence alignment results of GhNaKR3 protein and its homologs.

[0025] Figure 3 PEG 6000 After simulating drought stress treatment GhNaKR3 Figure showing the results of gene expression pattern analysis in cotton roots, stems, and leaves.

[0026] Figure 4 for GhNaKR3 Figure 1 shows the results of VIGS fragment amplification and TRV2::GhNaKR3 recombinant vector identification. In this figure, A represents... GhNaKR3 B represents the PCR amplification results of the VIGS gene fragment; B represents the PCR identification results of E. coli transformed with the TRV2::GhNaKR3 recombinant vector.

[0027] Figure 5 for GhNaKR3 Figure 1 shows the results of the VIGS gene silencing system and silencing efficiency test. In Figure 2, A represents the albino phenotype of TRV2::CLA1 positive control plants; and B represents the phenotype of TRV2::00 control plants and TRV2::GhNaKR3 silent plants. GhNaKR3 Results of gene relative expression level detection.

[0028] Figure 6 Phenotypic diagrams of TRV2::00 control plants and TRV2::GhNaKR3 silent plants under normal watering and natural drought stress conditions.

[0029] Figure 7 The figure shows the results of physiological index measurements of TRV2::00 control plants and TRV2::GhNaKR3 silent plants under normal watering and natural drought stress conditions. In the figure, A is the result of malondialdehyde (MDA) content measurement; B is the result of catalase (CAT) activity measurement; and C is the result of chlorophyll content measurement.

[0030] Figure 8 The image shows the pCAMBIA1302::GhNaKR3 recombinant expression vector.

[0031] Figure 9 for GhNaKR3The diagram shows the genetic transformation identification results of Arabidopsis thaliana overexpressing the gene. A is a schematic diagram of the T-DNA region structure of the pCAMBIA1302::GhNaKR3 recombinant expression vector; B shows the PCR identification results of the transgenic Arabidopsis thaliana; C shows the genetic transformation results of the transgenic Arabidopsis thaliana. GhNaKR3 Results of gene relative expression level detection.

[0032] Figure 10 Wild-type Arabidopsis thaliana and mannitol were cultured under normal conditions and simulated drought stress. GhNaKR3 Root growth phenotypes of Arabidopsis thaliana with gene overexpression, where A represents the root growth phenotype under normal 1 / 2 MS medium conditions; and B represents the root growth phenotype under 1 / 2 MS medium conditions containing 200 mmol / L mannitol.

[0033] Figure 11 Wild-type Arabidopsis thaliana and wild-type Arabidopsis thaliana under normal irrigation and natural drought stress conditions GhNaKR3 Phenotypic diagram of Arabidopsis thaliana plants with overexpressed genes.

[0034] Figure 12 Wild-type Arabidopsis thaliana and wild-type Arabidopsis thaliana under normal irrigation and natural drought stress conditions GhNaKR3 The figure shows the results of physiological index determination in Arabidopsis thaliana with gene overexpression. In the figure, A represents the result of malondialdehyde (MDA) content determination; B represents the result of catalase (CAT) activity determination; and C represents the result of chlorophyll content determination. Detailed Implementation

[0035] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0036] In this invention, GhNaKR3 The CDS sequence of the gene is shown in SEQ ID NO: 1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO: 2. Test materials

[0037] (1) Plant materials The cotton material used was Kezi cotton 312. Kezi cotton 312 seeds were soaked in water for 2 days. After germination, the seeds were planted in a culture medium with a vermiculite to nutrient soil volume ratio of 1:1 and cultured under conditions of 25±3℃, 50% relative humidity, and 16 h light / 8 h darkness.

[0038] The Arabidopsis thaliana material was wild-type Arabidopsis thaliana with a Colombian genetic background. Arabidopsis thalianaCol).

[0039] (2) Strains and plasmids The competent Escherichia coli cells were Trellef 5α, purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0040] The competent Agrobacterium tumefaciens cells used for the cotton virus-induced gene silencing assay were EHA105, purchased from Beijing Qingke Biotechnology Co., Ltd., and stored at -80℃.

[0041] The Agrobacterium tumefaciens competent cells used for Arabidopsis thaliana genetic transformation were GV3101, purchased from Beijing Qingke Biotechnology Co., Ltd., and stored at -80℃.

[0042] The vectors pTRV1, pTRV2, and the positive control vector pTRV2-CLA1 used in the cotton VIGS assay were all provided by the Institute of Biotechnology, Chinese Academy of Agricultural Sciences. Information regarding these vectors has been published in Chinese Invention Patent CN117535311B, "Upland Cotton GhCRP21 Gene and its Encoded Protein and Applications." Specifically, the empty pTRV2 vector is designated TRV2::00 in subsequent experiments and accompanying figures; the pTRV2-CLA1 positive control vector is designated TRV2::CLA1; and the recombinant vector obtained by ligating the GhNaKR3 gene VIGS fragment into the pTRV2 vector is designated TRV2::GhNaKR3.

[0043] The plant overexpression vector was pCAMBIA1302, provided by the Institute of Biotechnology, Chinese Academy of Agricultural Sciences.

[0044] (3) Main reagents RNA extraction was performed using the RNA Easy Fast Plant Tissue Kit; qRT-PCR was performed using the Taq ProUniversal SYBR qPCR Master Mix; PCR amplification was performed using 2×Phanta Flash Master Mix; PCR product recovery was performed using the TIANquick Midi Purification Kit; plasmid extraction was performed using the TIANprep Mini Plasmid Kit; and seamless ligation was performed using 2×Uniclone SeamLess Cloning Mix.

[0045] The reverse transcription kit was HiScript IV All-in-One Ultra RT SuperMix for qPCR, purchased from Novizan.

[0046] The malondialdehyde and catalase detection kits were purchased from Addison, and the chlorophyll detection kits were purchased from Solarbio.

[0047] (4) Statistical analysis Each experiment was performed in triplicate. Results are expressed as mean ± standard deviation. * indicates... P <0.05, ** indicates P <0.01, *** indicates P <0.001.

[0048] Example 1: Bioinformatics Analysis of the GhNaKR3 Gene 1.1 Bioinformatics analysis of the GhNaKR3 gene The amino acid sequence of GhNaKR3 in upland cotton was obtained using the upland cotton database in Phytozome (https: / / phytozome-next.jgi.doe.gov / ).

[0049] The physicochemical properties of the gene-encoded protein were analyzed using the online website ProtParam (http: / / web.expasy.org / protparam / ). The tertiary structure of the protein was analyzed using the Swiss Model website (https: / / www.swissmodel.expasy.org / ). A phylogenetic tree of GhNaKR3 was constructed using MEGA software and analyzed... https: / / espript.ibcp.fr / ESPript / cgi-bin / ESPript.cgi Visualization of amino acid sequence alignment on an online website.

[0050] 1.2 Physicochemical Properties Analysis of GhNaKR3 Protein The physicochemical properties of the GhNaKR3 protein were analyzed. It has a protein length of 223 amino acids, an isoelectric point (pI) of 9.92, and a molecular weight of approximately 24.371 kDa. The average hydrophilicity index of GhNaKR3 is -0.513, indicating that it is not a hydrophobic protein but rather tends to be hydrophilic. The instability index of GhNaKR3 protein is 62.54, suggesting a certain degree of instability. Specific results are shown in Table 1.

[0051] Table 1 Physicochemical properties of GhNaKR3 protein

[0052] 1.3 GhNaKR3 protein structure analysis The predicted tertiary structure of the GhNaKR3 protein is as follows: Figure 1 As shown.

[0053] Structural prediction results show that the GhNaKR3 protein as a whole has many flexible regions without a fixed spatial conformation, with local segments folding to form relatively conserved HMA domains. These HMA domains consist of an α / β hybrid folding backbone composed of α-helices, antiparallel β-sheets, and flexible loop regions.

[0054] Structural localization analysis revealed two conserved cysteine ​​residues, Cys154 and Cys157, within the HMA domain, which may be related to the redox response and metal ion binding function of the domain.

[0055] 1.4 Phylogenetic analysis and amino acid sequence alignment of GhNaKR3 protein To analyze the evolutionary relationship of GhNaKR3 in the NaKR family of upland cotton, a phylogenetic tree was constructed using NaKR homologous protein sequences from plants including Arabidopsis thaliana, hibiscus, apple, tobacco, cucumber, rice, sorghum, millet, wheat, and maize. The results are as follows: Figure 2 As shown in A.

[0056] The results showed that GhNaKR3 was most closely related to HsNaKR3 and clustered with MdNaKR2, NtNaKR1 and CucNaKR1; GhNaKR3 was in a different branch from its homologous proteins such as OsNaKR3 and TaNaKR2 from monocotyledonous plants.

[0057] The amino acid sequence alignment results are as follows Figure 2 As shown in Figure B. The results indicate that the HMA domain of GhNaKR3 and its homologs is highly conserved, suggesting that the HMA domain may be an important structural basis for the function of NaKR family proteins.

[0058] Example 2 GhNaKR3 Gene cloning and VIGS validation of its function 2.1 Extraction of total RNA from cotton and synthesis of cDNA Once the Kezi cotton 312 plants have grown to the four-leaf stage, use PEG at a weight / volume percentage of 20%. 6000 The solution was subjected to simulated drought stress treatment, and cotton root, stem and leaf tissues were collected at 0 h, 1 h, 3 h, 6 h, 12 h and 24 h after treatment.

[0059] Samples were immediately frozen in liquid nitrogen after collection, and total RNA was extracted according to the instructions of the RNA Easy Fast Plant Tissue Kit. After RNA extraction, the RNA concentration was detected using an ultra-micro spectrophotometer, followed by reverse transcription to synthesize cDNA. The remaining RNA was stored at -80℃.

[0060] 2.2 qRT-PCR analysis qRT-PCR analysis was performed using Taq Pro Universal SYBR qPCR Master Mix reagents, with three biological replicates per group, using cotton... UBQ The gene is an internal control, based on 2 -ΔΔCt The method calculates the relative expression level of genes.

[0061] Designed using Primer Premier 5 GhNaKR3 Gene-specific primers, primer sequences are as follows: Upstream primer: 5′-CATTCGTTCCTTCTCAGCC-3′, as shown in SEQ ID NO: 3; Downstream primer: 5′-TTTTCCTTCACATCCTTTGC-3′, as shown in SEQ ID NO: 4.

[0062] cotton UBQ The upstream primer sequence of the internal reference gene is 5′-GCAAGTGTTGGGTTCAAAGCTGGTG-3′, as shown in SEQ ID NO: 10; the downstream primer sequence is 5′-CCAGGTTGAGGAGTTACTCGGAATGCTG-3′, as shown in SEQ ID NO: 11.

[0063] The qRT-PCR reaction system is shown in Table 2, and the qRT-PCR reaction conditions are shown in Table 3.

[0064] Table 2 qRT-PCR reaction system

[0065] Table 3 qRT-PCR reaction conditions

[0066] 2.3 GhNaKR3 Gene cloning and product recovery according to GhNaKR3 The CDS sequence of the gene was used to design VIGS-specific primers (F: gtgagtaaggttaccgaattcTCTAGCGTGGACCATCGCTC, as shown in SEQ ID NO: 5; R: cgtgagctcggtaccggatccCCACCCTCAAAACCACCACC, as shown in SEQ ID NO: 6) to amplify the specific VIGS fragment. The PCR amplification system is shown in Table 4 below, and the PCR reaction procedure is shown in Table 5 below. Some operations were performed on ice.

[0067] The nucleotide sequence of the VIGS fragment is shown in SEQ ID NO: 7, corresponding to positions 52 to 451 of the CDS sequence of the GhNaKR3 gene shown in SEQ ID NO: 1.

[0068] Table 4 PCR amplification reaction system

[0069] The PCR amplification procedure is shown in Table 5.

[0070] Table 5 PCR amplification reaction procedure

[0071] Take 5 μL of PCR product, detect the target band by 1% agarose gel electrophoresis, and recover the PCR product using TIANquick MidiPurification Kit.

[0072] After PCR product recovery, 1 μL of the recovered product was taken, and the concentration and purity of the DNA fragments were determined using an ultra-micro spectrophotometer. The DNA was analyzed at OD... 260 It has a distinct absorption peak at OD 260 / OD 280 When the ratio is 1.7 to 1.9, the recovered DNA fragments are stored at -20°C for later use.

[0073] 2.4 Plasmid digestion, recovery, and ligation The multiple cloning sites of the pTRV2 vector were analyzed, and the pTRV2 vector was double-digested using EcoRI-HF and BamHI-HF.

[0074] The enzyme-digested linearized pTRV2 vector and GhNaKR3 The VIGS gene fragment was ligated using a seamless cloning method, and the ligation system is shown in Table 6.

[0075] Table 6 Connection System

[0076] After ligation, the TRV2::GhNaKR3 recombinant vector was obtained.

[0077] 2.5 Transformation of Escherichia coli with recombinant vector and identification of positive clones The ligation product from section 2.4 was transformed into Trellef 5α Escherichia coli competent cells, following the instructions for use of competent cells.

[0078] After successful transformation, single clones were selected for bacterial culture PCR identification, and the clones that were identified as positive were sequenced.

[0079] 2.6 Plasmid Extraction Select positive clones with correct sequencing results for amplification culture, and extract the TRV2::GhNaKR3 recombinant vector according to the TIANprep Mini Plasmid Kit instructions.

[0080] The concentration and purity of the plasmid were determined using an ultra-micro spectrophotometer. The plasmid DNA was analyzed at OD... 260 It has a distinct absorption peak at OD 260 / OD 280 When the ratio is 1.7 to 1.9, the plasmid should be stored at -20°C for later use.

[0081] 2.7 Transformation of Agrobacterium tumefaciens EHA105 with recombinant vector The TRV2::GhNaKR3 recombinant vector was transformed into Agrobacterium tumefaciens EHA105 using the freeze-thaw method. The transformation operation was carried out in a clean bench.

[0082] After single colonies have grown on the plate, select colonies for bacterial culture PCR identification. Select Agrobacterium tumefaciens cultures with correct identification results for further culture and preservation in glycerol at -80℃.

[0083] 2.8 VIGS Test (1) Activation of strains: Agrobacterium tumefaciens EHA105 bacterial suspensions stored at -80°C carrying TRV2::00, TRV2::GhNaKR3, TRV2::CLA1 and pTRV1 vectors respectively were inoculated into LB liquid medium containing 50 μg / mL kanamycin and 50 μg / mL rifampin and activated at 28°C and 200 r / min.

[0084] (2) The activated bacterial culture was added to LB liquid medium containing 50 μg / mL Kan and 50 μg / mL Rif (this medium contains 10 mM MES pH=6.5 and 20 μM AS) and cultured at 28℃ and 200 r / min until OD. 600 =0.8~1.0.

[0085] (3) Centrifuge at 4℃ and 6000 r / min for 10 min.

[0086] (4) Discard the supernatant and retain the precipitated bacteria, resuspend in sterile water, and centrifuge twice.

[0087] (5) Resuspend Agrobacterium cells in osmotic buffer (containing 10 mM MgCl2, 10 mM MES, pH = 5.6, and 20 μM acetylsuccinone) until OD. 600 =1.0.

[0088] (6) Let stand at room temperature for 3 hours.

[0089] (7) Before infection, the TRV2::00, TRV2::GhNaKR3 or TRV2::CLA1 strain suspensions resuspended in buffer solution are mixed with the pTRV1 strain suspension in equal volumes.

[0090] (8) Inoculation with Agrobacterium tumefaciens solution: The leaf syringe immersion method was used. Cotton seedlings with good growth and only two cotyledons were selected. First, the back of the cotyledon was gently punctured with the needle of a syringe to create a small wound, without piercing the leaf. Then, the bacterial solution was injected into the wound on the back of the leaf using a syringe without the needle, so that both cotyledons were completely saturated. Untreated plants were used as controls.

[0091] The types of injectable plants are as follows: The pTRV1+TRV2::00 group served as a negative control.

[0092] pTRV1+TRV2::CLA1 group: as a positive control, the appearance of white phenotype in the plants indicates that the VIGS system is functioning normally; pTRV1+TRV2::GhNaKR3 group, as GhNaKR3 Gene silencing group.

[0093] To maintain consistency with the experimental results and figure labels, the three treatment groups will be referred to as TRV2::00 group, TRV2::CLA1 group, and TRV2::GhNaKR3 group, respectively.

[0094] (9) Cultivation of plants after gene silencing: After gene silencing, the plants were first cultured in the dark at 23°C for 24 h, and then cultured for 14 days under a 16 h / 8 h light / dark cycle and a temperature of 23°C. The albino phenotype was observed and the gene silencing efficiency was detected. After effective silencing, the plants were subjected to natural drought for 7 days. The phenotypes of the control and silent plants were observed, and relevant drought physiological indicators were detected.

[0095] 2.9 qRT-PCR detection of silencing efficiency qRT-PCR was used to detect TRV2::GhNaKR3 silencing group GhNaKR3 The gene silencing efficiency was measured, with the TRV2::00 group as a control. The detection method is described in Section 2.2 of this embodiment.

[0096] 2.10 Measurement of physiological indicators under drought stress (1) Determination of malondialdehyde content Leaves were collected from TRV2::00 control plants and TRV2::GhNaKR3 silent plants after 7 days of normal watering and natural drought treatment, respectively. The malondialdehyde (MDA) content was measured using the Addison Company's malondialdehyde (MDA) detection kit and calculated based on the fresh weight of the samples.

[0097] The malondialdehyde (MDA) content is calculated using the following formula: Malondialdehyde content (nmol / g fresh weight) = [ΔA ÷ (ε × d) × V² × 10⁹] ÷ (W × V₁ ÷ V) = 32.3 × ΔA ÷ W Where V is the total volume of the sample extract (1 mL); V1 is the volume of sample added to the reaction system (0.2 mL); and V2 is the total reaction volume of the sample added and the working solution (5 × 10⁻⁶). -4 L; d is the optical path length, 0.5 cm; ε is the molar extinction coefficient of MDA, 155 × 10⁻⁶. 3 L / mol / cm; W is the sample mass, in g.

[0098] (2) Determination of CAT activity Leaves were collected from TRV2::00 control plants and TRV2::GhNaKR3 silent plants after 7 days of normal watering and natural drought treatment. The catalase activity was measured using the Addison catalase assay kit and calculated based on the fresh weight of the samples.

[0099] Enzyme activity unit definition: At 25°C, one enzyme activity unit (U) is defined as the ability of one gram of tissue to catalyze the decomposition of 1 μmol H2O2 per minute.

[0100] CAT(μmol / min / g)=[(ΔA+0.0135)÷0.1413]÷(W×V1÷V)÷T×D=141.54×(ΔA+0.0135)÷W×D Where ΔA = A blank - A determination; V is the volume of extract added, 1 mL; V1 is the volume of sample added, 0.01 mL; T is the reaction time, 5 min; W is the sample mass, g; D is the dilution factor, which is 1 for undiluted samples.

[0101] (3) Determination of chlorophyll content Leaves were collected from TRV2::00 control plants and TRV2::GhNaKR3 silent plants after 7 days of normal watering and natural drought treatment, respectively. Chlorophyll content was measured using a Solarbio chlorophyll assay kit. Total chlorophyll content was calculated using the following formula: Total chlorophyll content (mg / g fresh weight) = (33.7 × A645 + 13.4 × A663) × V_extracted × F ÷ W ÷ 1000 Where V is the extraction volume of 1.5 mL; F is the dilution factor; and W is the sample mass in g.

[0102] The measurement results are as follows Figure 7As shown. Under normal watering conditions, there were no significant differences in MDA content, CAT activity, and chlorophyll content between TRV2::GhNaKR3 silenced plants and TRV2::00 control plants. After natural drought treatment, the MDA content of TRV2::GhNaKR3 silenced plants was significantly higher than that of TRV2::00 control plants, while CAT activity and chlorophyll content were significantly lower. These results indicate that silencing... GhNaKR3 Genes can reduce the drought resistance of cotton.

[0103] 2.11 GhNaKR3 Gene expression pattern analysis To investigate GhNaKR3 To investigate gene expression characteristics in cotton under drought stress, cotton was treated with 20% PEG6000 to simulate drought stress. Root, stem, and leaf tissues were collected at 0 h, 1 h, 3 h, 6 h, 12 h, and 24 h after treatment. Total RNA was extracted and cDNA was obtained by reverse transcription. qRT-PCR was used for detection. GhNaKR3 The relative expression level of genes.

[0104] The results are as follows Figure 3 As shown, GhNaKR3 The gene was induced 1 h after drought stress treatment, with high expression levels in roots, reaching a peak at 12 h after treatment, indicating that... GhNaKR3 Genes can respond to drought stress in cotton.

[0105] 2.12 GhNaKR3 Construction of the VIGS gene vector In construction GhNaKR3 When using a VIGS vector for gene delivery, the first step is to... GhNaKR3 The CDS sequence of the gene was analyzed, and a specific fragment was selected as the VIGS fragment. Based on the sequence analysis results, the fragments selected for the VIGS assay were... GhNaKR3 The gene fragment is 400 bp in size, and its nucleotide sequence is shown in SEQ ID NO: 7.

[0106] Using cDNA from *Cotton Cocos* 312 as a template, the VIGS fragment of the GhNaKR3 gene was amplified using the VIGS-specific primers shown in SEQ ID NO: 5 and SEQ ID NO: 6. Electrophoresis results are shown below. Figure 4 As shown in A, the amplified product size is 400 bp, which is consistent with the expected result.

[0107] The pTRV2 vector was double-digested with EcoRI-HF and BamHI-HF to recover the linearized vector. The linearized pTRV2 vector was ligated with the VIGS fragment of the GhNaKR3 gene using a seamless cloning method to obtain the TRV2::GhNaKR3 recombinant vector. The ligation product was then transformed into Trelleb 5α competent Escherichia coli cells.

[0108] After single colonies grew on the plate, they were picked and identified by PCR using the VIGS-specific primers shown in SEQ ID NO: 5 and SEQ ID NO: 6. The electrophoresis results are as follows: Figure 4 As shown in B, the target band size of the positive clone is 400 bp, which is consistent with the expected result.

[0109] Take 150 μL of the positive clone culture and send it for sequencing. Select the clones with correct sequencing results for amplification culture, strain preservation, and plasmid extraction.

[0110] The extracted TRV2::GhNaKR3 recombinant vector was transformed into Agrobacterium tumefaciens EHA105. After single colonies grew on the plates, they were picked and identified by VIGS-specific primers using PCR. Agarose gel electrophoresis showed that the target band was 400 bp in size, consistent with the results of PCR identification after transformation of Escherichia coli.

[0111] Select Agrobacterium tumefaciens bacterial suspension with correct identification results, inoculate it in LB liquid medium containing kanamycin and rifampin and culture overnight, preserve the bacterial suspension with glycerol and store at -80℃ to complete the construction of TRV2::GhNaKR3 recombinant vector and Agrobacterium tumefaciens transformation.

[0112] 2.13 GhNaKR3 Gene silencing efficiency test Two weeks after injection of Agrobacterium tumefaciens solution, the true leaves of TRV2::CLA1 positive control plants showed leukoplakia, such as... Figure 5 As shown in A, this indicates that the VIGS system is operating normally.

[0113] Select respectively GhNaKR3 Ten gene-silenced plants and ten pTRV2 empty vector control plants were used. Total RNA was extracted from the leaves and detected by qRT-PCR. GhNaKR3 The efficiency of gene silencing.

[0114] The results are as follows Figure 5 As shown in B, compared with the TRV2::00 control plants, the TRV2::GhNaKR3 silent plants had [a higher concentration of these plants]. GhNaKR3 The relative expression level of the gene was significantly reduced, with a silencing efficiency of 82.5%, indicating that... GhNaKR3The gene was effectively silenced and can be used for subsequent drought stress treatment.

[0115] 2.14 GhNaKR3 Drought resistance phenotype analysis of gene-silenced cotton right GhNaKR3 VIGS silencing of the gene yielded the following results: Figure 6 As shown.

[0116] Under normal watering conditions, there was no significant difference in growth status between TRV2::GhNaKR3 silent plants and TRV2::00 control plants.

[0117] After 7 days of natural drought treatment, compared with the TRV2::00 control plants, the TRV2::GhNaKR3 silent plants showed more severe wilting symptoms, with leaves exhibiting water loss, wrinkling, and brittleness.

[0118] The results of relevant physiological index measurements are as follows: Figure 7 As shown. Under normal watering conditions, there were no significant differences in MDA content, CAT activity, and chlorophyll content between TRV2::GhNaKR3 silent plants and TRV2::00 control plants. After natural drought treatment, the MDA content of TRV2::GhNaKR3 silent plants was significantly higher than that of TRV2::00 control plants, while CAT activity and chlorophyll content were significantly lower than those of TRV2::00 control plants.

[0119] The above results further indicate that silencing the GhNaKR3 gene reduces the drought resistance of cotton.

[0120] Example 3 (Transfer) GhNaKR3 Construction and drought resistance verification of Arabidopsis thaliana with gene overexpression 3.1 GhNaKR3 Amplification and recovery of gene CDS fragments according to GhNaKR3 Primers were designed based on the CDS sequence of the gene. The primer sequences are as follows: 1302-GhNaKR3-F: 5′-ggactcttgaccatggATGAAAGGAATGGATCTTTTTTG-3′, as shown in SEQ ID NO: 8; 1302-GhNaKR3-R: 5′-gtcagatctaccatggGTAATTCATTTTGACCATTGGAGA-3′, as shown in SEQ ID NO: 9.

[0121] As shown in SEQ ID NO: 1 GhNaKR3 The 3' terminator codon of the gene's CDS sequence is TGA. To achieve...GhNaKR3 Fusion expression with mGFP5 was achieved using 1302-GhNaKR3-F and 1302-GhNaKR3-R amplification of the expression shown in SEQ ID NO: 1. GhNaKR3 The sequence in the gene CDS sequence excluding the 3′ stop codon TGA.

[0122] Using cotton cDNA as a template, the above primers were used for amplification. GhNaKR3 The CDS fragment of the gene was extracted, and the PCR product was recovered. The PCR amplification and product recovery methods were as described in Section 2.3 of Example 2. The annealing temperature was determined based on the Tm values ​​of the primers shown in SEQ ID NO: 8 and SEQ ID NO: 9.

[0123] 3.2 Construction of the pCAMBIA1302::GhNaKR3 recombinant expression vector The multiple cloning sites of the pCAMBIA1302 vector were analyzed. The pCAMBIA1302 vector was digested with NcoI and the linearized vector was recovered.

[0124] Using a seamless cloning method to remove the stop codon GhNaKR3 The encoded sequence fragment is ligated to the linearized pCAMBIA1302 vector, so that... GhNaKR3 The coding sequence is in the same reading frame as the mGFP5 coding sequence in the vector, resulting in the pCAMBIA1302::GhNaKR3 recombinant expression vector for fusion expression of GhNaKR3-mGFP5 protein. Its vector map is shown below. Figure 8 As shown.

[0125] 3.3 Transformation of recombinant plasmids into competent E. coli cells and identification of monoclonal cells The recombinant plasmid was transformed into Trellef 5α competent E. coli cells, following the instructions for use of the competent cells from Novizan Biosciences Co., Ltd. After successful transformation, single clones were selected for PCR identification, and clones that tested positive by PCR were sent for sequencing.

[0126] 3.4 Plasmid Extraction Plasmid extraction was performed according to the TIANprep Mini Plasmid Kit instructions. The concentration and purity of the extracted plasmids were determined using a micro spectrophotometer. The plasmid DNA should be extracted at an OD value of [insert value here]. 260 There is a significant absorption peak at OD. 260 / OD 280 The ratio should be between 1.7 and 1.9.

[0127] 3.5 Plasmid transformation of Agrobacterium competent cells Transform the pCAMBIA1302::GhNaKR3 plasmid into GV3101 competent cells according to the manufacturer's instructions. Spread the plasmid on LB solid medium containing Kan and Rif and culture in a shaker at 28°C for 2–3 days. Select single clones for identification.

[0128] 3.6 Arabidopsis genetic transformation (1) Agrobacterium infection Agrobacterium was picked up in a resuspension to prepare OD. 600 Prepare a Agrobacterium resuspension at a concentration of 0.8–1.2, add Silwet-77 to a concentration of 0.02%, dip all the inflorescences of Arabidopsis thaliana material into the bacterial solution for 2–3 seconds, seal with a film to maintain humidity >90%, and incubate in the dark at 25°C for 24 hours. The infection cycle is 7 days, with a total of 3 infection cycles.

[0129] (2) Harvesting After soaking, the seedlings were placed at 23℃ for 16 h / 8 h light / dark incubation until they produced seeds. The mature pods were gently rubbed onto clean white paper, wrapped, and dried at 37℃ for 24 h. After drying, the seeds were sieved through a 60-mesh sieve and stored at 4℃.

[0130] (3) Disinfection and screening The harvested T1 generation seeds were disinfected with 95% ethanol for 10 min, then with 75% ethanol for 10 min, and washed 2–3 times with sterile water (1 min each time). The seeds were then evenly spread on 1 / 2 MS selection medium containing 35 mg / L hygromycin B and incubated at 4°C for 2–3 days. The plates were then removed and incubated at 23–25°C under a 16 h / 8 h light / dark cycle for 10–14 days. The surviving T1 seedlings were transplanted into nutrient soil and incubated at 23°C under a 16 h / 8 h light / dark cycle. After approximately 20 days of growth, Arabidopsis thaliana genomic DNA was extracted for PCR detection.

[0131] The upstream primer sequence for PCR identification was 5′-CTGCCCGCTGTTCTACAACCGG-3′, as shown in SEQ ID NO: 12; the downstream primer sequence was 5′-GGAGCATATACGCCCGGAGTC-3′, as shown in SEQ ID NO: 13; the expected amplification product length was 500 bp.

[0132] (4) Screening of T2 generation seeds T1 generation plants that tested positive by PCR were individually harvested to obtain T2 generation seeds. The T2 generation seeds were then disinfected and screened for hygromycin resistance using the same method. The surviving T2 generation seedlings were transplanted into nutrient soil, maintaining the same lineage number as the corresponding T1 generation plant. After approximately 20 days of growth, Arabidopsis thaliana genomic DNA was extracted for PCR testing to obtain positive T2 generation plants, which were then used for subsequent... GhNaKR3 Gene expression levels and drought resistance function were detected.

[0133] 3.7 Determination of drought stress indices The methods for determining the MDA content, CAT activity, and chlorophyll content of Arabidopsis leaves are as described in Section 2.10 of Example 2.

[0134] 3.8 GhNaKR3 Genetic transformation identification of Arabidopsis thaliana with overexpression of genes Will GhNaKR3 The gene was transferred into Arabidopsis thaliana to further verify its drought resistance function. The pCAMBIA1302::GhNaKR3 recombinant expression vector was constructed using the above method, and its complete vector map is shown below. Figure 8 As shown in the diagram, the T-DNA region structure is as follows: Figure 9 As shown in A, wild-type Arabidopsis thaliana was genetically transformed using the inflorescence staining method.

[0135] After screening T1 generation seeds on 1 / 2 MS medium containing 35 mg / L hygromycin B, resistant and positive seedlings were transplanted into soil for culture; PCR detection was performed on the T1 generation plants to obtain positive lines, and the results are as follows. Figure 9 As shown in B in the diagram.

[0136] QRT-qPCR was performed on T2 generation positive lines for analysis. GhNaKR3 The relative expression level of genes.

[0137] qRT-PCR with Arabidopsis thaliana Atactin2 The gene is an internal reference gene. The upstream primer sequence of the internal reference gene is 5′-TCCATGAAACAACTTACAACTCCA-3′, as shown in SEQ ID NO: 14, and the downstream primer sequence is 5′-CGTACTCACTCTTTGAAATCCACA-3′, as shown in SEQ ID NO: 15. GhNaKR3 The gene detection used primers shown in SEQ ID NO:3 and SEQ ID NO:4, and the reaction system and reaction conditions were as described in Section 2.2 of Example 2.

[0138] The results are as follows Figure 9As shown in C, the relative expression levels of the GhNaKR3 gene were high in the OE2, OE3, and OE5 lines. The OE2, OE3, and OE5 lines were selected for further verification of drought resistance function.

[0139] 3.9 Overexpression GhNaKR3 Arabidopsis thaliana culture medium phenotype To investigate GhNaKR3 The function of genes in drought stress response was investigated by adding 200 mmol / L mannitol to 1 / 2 MS medium to simulate drought stress, and comparing wild-type Arabidopsis and wild-type Arabidopsis after 14 days of culture. GhNaKR3 Growth phenotypes of gene overexpression lines OE2, OE3, and OE5.

[0140] The results are as follows Figure 10 As shown. On normal 1 / 2 MS medium, wild-type Arabidopsis thaliana and various... GhNaKR3 There were no significant differences in the aboveground and root growth of the gene-overexpressing lines.

[0141] On 1 / 2 MS medium containing 200 mmol / L mannitol, the growth of wild-type Arabidopsis thaliana was significantly inhibited, as evidenced by a shortened taproot length and a reduced number of lateral roots. GhNaKR3 The growth of the gene-overexpressing lines was significantly reduced, and their root systems were more developed.

[0142] 3.10 Overexpression GhNaKR3 Arabidopsis thaliana drought resistance phenotype Wild-type Arabidopsis thaliana with consistent growth status and GhNaKR3 Gene overexpression lines OE2, OE3, and OE5 were divided into a normal watering group and a natural drought group, respectively. Watering was stopped for 7 days in the natural drought group, while the normal watering group maintained normal water management, and all other culture conditions were the same.

[0143] Under normal watering conditions, wild-type Arabidopsis thaliana and GhNaKR3 There was no significant difference in the growth status of Arabidopsis thaliana with gene overexpression, and the leaves remained healthy and fully extended.

[0144] After 7 days of natural drought treatment, wild-type Arabidopsis thaliana showed obvious symptoms such as wilting, yellowing and drying of leaves; GhNaKR3 The Arabidopsis thaliana with overexpression of the gene showed less wilting and significantly less damage from drought stress than the wild-type Arabidopsis thaliana, as shown in the results. Figure 11 As shown.

[0145] The above phenotypic results indicate that GhNaKR3 Overexpression of the gene can enhance the tolerance of Arabidopsis thaliana to drought stress. GhNaKR3 Genes are positive regulators that regulate drought resistance in plants.

[0146] 3.11 OverexpressionGhNaKR3 Determination of drought stress indices in Arabidopsis thaliana Wild-type Arabidopsis thaliana after normal watering and natural drought treatment and GhNaKR3 MDA content, CAT activity, and chlorophyll content were measured in Arabidopsis thaliana with gene overexpression.

[0147] The results are as follows Figure 12 As shown. Under normal watering conditions, GhNaKR3 The MDA content, CAT activity, and chlorophyll content of gene-overexpressing Arabidopsis thaliana were not significantly different from those of wild-type Arabidopsis thaliana.

[0148] After drought stress, GhNaKR3 The MDA content of Arabidopsis thaliana with gene overexpression was significantly lower than that of wild-type Arabidopsis thaliana, while CAT activity and chlorophyll content were significantly higher, further indicating that... GhNaKR3 Overexpression of the gene can enhance the drought resistance of Arabidopsis thaliana.

Claims

1. Cotton GhNaKR3 The application of genes in improving plant drought resistance is characterized by, The GhNaKR3 The CDS sequence of the gene is shown in SEQ ID NO: 1; the application is achieved by overexpressing the gene in plants. GhNaKR3 Genetic implementation; the plant in question is Arabidopsis thaliana.

2. The application according to claim 1, characterized in that, The overexpression was achieved using the pCAMBIA1302::GhNaKR3 recombinant expression vector; the pCAMBIA1302::GhNaKR3 recombinant expression vector was expressed by the vector shown in SEQ ID NO:

1. GhNaKR3 The coding sequence in the gene CDS sequence, except for the 3′ stop codon TGA, was ligated into the pCAMBIA1302 vector and then ligated in the same frame with the mGFP5 coding sequence in the pCAMBIA1302 vector.

3. The application according to claim 2, characterized in that, The pCAMBIA1302::GhNaKR3 recombinant expression vector was transformed into Agrobacterium tumefaciens GV3101, and Arabidopsis thaliana was transformed by inflorescence immersion using Agrobacterium tumefaciens GV3101 carrying the pCAMBIA1302::GhNaKR3 recombinant expression vector.

4. A method for improving the drought resistance of Arabidopsis thaliana, characterized in that, Includes the following steps: (1) The SEQ ID NO: 1 shown GhNaKR3 The coding sequence in the gene CDS sequence, except for the 3′ stop codon TGA, was ligated into the pCAMBIA1302 vector and co-linked with the mGFP5 coding sequence in the pCAMBIA1302 vector to obtain the pCAMBIA1302::GhNaKR3 recombinant expression vector. (2) The pCAMBIA1302::GhNaKR3 recombinant expression vector was transformed into Agrobacterium tumefaciens GV3101; (3) Agrobacterium tumefaciens GV3101 carrying the pCAMBIA1302::GhNaKR3 recombinant expression vector was used to transform Arabidopsis thaliana by inflorescence infiltration method; (4) The Arabidopsis seeds obtained after transformation were screened for hygromycin resistance, and the screened plants were identified by PCR and the expression level of GhNaKR3 gene was detected to obtain overexpressing... GhNaKR3 Arabidopsis thaliana plants with genetic material.

5. A drought-resistant Arabidopsis thaliana, characterized in that, It is prepared by the method described in claim 4.

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