Application of ramie glutathione synthetase gene in regulating plant drought resistance, growth ability or antioxidant capacity

Abstract

The present invention belongs to the field of biotechnology applications and relates to the use of a ramie glutathione synthetase gene in regulating plant drought resistance. The sequence of the ramie glutathione synthetase gene is shown in SEQ ID NO. 1. Results from the present invention show that under drought stress, Arabidopsis thaliana overexpressing BnGS exhibited higher root length, biomass, sodium content, and potassium content than wild-type plants, and exhibited significantly higher antioxidant enzyme activities, while MDA, H2O2, and AsA content were significantly lower than those of wild-type plants. This indicates that overexpressing BnGS in Arabidopsis thaliana can enhance antioxidant capacity, eliminate excess free radicals, and improve plant drought resistance.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology application and relates to the application of ramie glutathione synthetase gene in regulating plant drought resistance, growth ability or antioxidant capacity. Background Art

[0002] Drought stress is a globally prevalent natural environmental stressor, severely impacting plant growth and development. Glutathione synthetase (GS) is a key enzyme in plant glutathione (GSH) synthesis. GSH synthesis requires two enzymes: γ-glutamate-cysteine ​​synthetase (γ-GCS) and glutathione synthetase (GS). γ-GCS catalyzes the ligation of glutamate and cysteine ​​to form γ-glutamylcysteine, while GS catalyzes the condensation of the carboxyl group of γ-glutamylcysteine ​​with the α-amino group of glycine to produce glutathione. Furthermore, prior art has revealed that glutathione S-transferase (GST) genes play an important role in plant drought resistance. However, the functional mechanisms and regulatory targets of GST and GSH synthase genes differ significantly.

[0003] At present, the research on glutathione synthetase mainly focuses on the following aspects:

[0004] 1. Researchers successfully cloned the potato glutathione synthetase (StGS) gene using homologous cloning, constructed overexpression of the gene, and screened, identified, and functionally verified StGS-overexpressing transgenic plants. The results showed that StGS is localized in potato leaves, roots, stem apical meristem, and mature stems. They found that StGS affects potato GSH synthesis and increases both above- and below-ground potato biomass. The results also confirmed that StGS can increase GSH content in two transgenic potato lines overexpressing the gene.

[0005] 2. Researchers cloned the glutathione synthetase gene fragment Rsgsh of rice sheath blight pathogen, and then used fluorescent quantitative PCR to collect and analyze samples at different time points after rice was inoculated with sheath blight pathogen. Through specific primer detection, they found that the expression pattern of the glutathione synthetase gene was closely related to the pathogen infection process. Expression analysis showed that the gene was induced to express at the early stage of infection and the expression level continued to increase over time, indicating that the gene may play an important role in the interaction between the pathogen and rice, participating in the pathogen infection process and possible defense mechanism.

[0006] 3. Researchers cloned and sequenced the Lycium barbarum GS gene. They then subjected Lycium barbarum seedlings of uniform growth to heavy metal and salt stress for varying durations to investigate changes in LcGS expression. The results showed that the LcGS gene's expression abundance varied under these stresses, with expression levels also varying at different treatment times. This suggests that the gene is involved in responses to these stresses, and overexpression of the LcGS gene may improve plant tolerance to these stresses. However, this Lycium barbarum gene is not present in ramie.

[0007] Ramie ( Boehmeria nivea ) is a perennial bast fiber crop native to China that exhibits strong resistance to abiotic stresses such as drought and heavy metal stress. Currently, there are no reports on whether glutathione synthetase can enhance a plant's ability to withstand drought. Summary of the Invention

[0008] The purpose of the present invention is to provide an application of a ramie glutathione synthetase gene in regulating plant drought resistance, growth ability or antioxidant activity.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] Application of a ramie glutathione synthetase gene in regulating plant drought resistance. The sequence of the ramie glutathione synthetase gene is shown in SEQ ID NO.1.

[0011] In a preferred embodiment, the application is: cloning the ramie glutathione synthetase gene and constructing an overexpression vector, and then transfecting the overexpression vector into wild-type plants to obtain ramie glutathione synthetase gene overexpressing plants, which are drought-tolerant plants.

[0012] In a preferred embodiment, the plants include ramie and Arabidopsis thaliana.

[0013] The use of a reagent for increasing the expression of ramie glutathione synthetase gene in the preparation of a reagent for regulating plant drought resistance, wherein the reagent for increasing the expression of ramie glutathione synthetase gene comprises one or more of the following:

[0014] A, primers for amplifying ramie glutathione synthetase gene;

[0015] B. a kit containing primers for amplifying the ramie glutathione synthetase gene;

[0016] C. a recombinant vector containing primers for amplifying the ramie glutathione synthetase gene;

[0017] D. Agrobacterium containing primers for amplifying the ramie glutathione synthetase gene.

[0018] In a preferred embodiment, the recombinant vector containing primers for amplifying ramie glutathione synthetase gene is pCAMBIA1300-35S-BnGS-myc.

[0019] In a preferred embodiment, the sequences of the primers for amplifying the ramie glutathione synthetase gene are shown as SEQ ID NO. 4 and SEQ ID NO. 5.

[0020] Use of a reagent for increasing the expression of ramie glutathione synthetase gene in the preparation of a reagent for regulating plant growth ability, wherein the reagent for increasing the expression of ramie glutathione synthetase gene comprises one or more of the following:

[0021] A, primers for amplifying ramie glutathione synthetase gene;

[0022] B. a kit containing primers for amplifying the ramie glutathione synthetase gene;

[0023] C. a recombinant vector containing primers for amplifying the ramie glutathione synthetase gene;

[0024] D. Agrobacterium containing the recombinant vector.

[0025] In a preferred embodiment, the plant growth capacity includes root length, biomass, sodium content and potassium content.

[0026] Use of a reagent for increasing the expression of ramie glutathione synthetase gene in the preparation of a reagent for regulating plant antioxidant activity, wherein the reagent for increasing the expression of ramie glutathione synthetase gene comprises one or more of the following:

[0027] A, primers for amplifying ramie glutathione synthetase gene;

[0028] B. a kit containing primers for amplifying the ramie glutathione synthetase gene;

[0029] C. a recombinant vector containing primers for amplifying the ramie glutathione synthetase gene;

[0030] D. Agrobacterium containing primers for amplifying the ramie glutathione synthetase gene.

[0031] In a preferred embodiment, the regulation is increasing.

[0032] Based on the same inventive concept, the present invention also requires a method for constructing drought-resistant ramie, specifically:

[0033] The ramie glutathione synthetase gene is cloned and an overexpression vector is constructed, and then the overexpression vector is transfected into wild-type ramie through transgenic technology to obtain ramie glutathione synthetase gene overexpression plants, which are drought-tolerant ramie.

[0034] In the present invention, under drought stress, Arabidopsis plants overexpressing BnGS showed higher root length, biomass, and sodium and potassium contents than the wild type, and exhibited significant advantages in antioxidant enzyme activity, while MDA, H₂O₂, and AsA contents were significantly lower than those of the wild type. This suggests that overexpressing BnGS in Arabidopsis can enhance antioxidant capacity, eliminate excess free radicals, and improve plant drought resistance.

[0035] The beneficial effects of the present invention are:

[0036] The present invention determined the main metabolic pathways and related genes in ramie in response to drought stress through statistical analysis of experimental results. Combining transgenic technology and physiological and biochemical experiments, it revealed the synergistic effects of BnGS through GSH metabolism, ROS scavenging and activation of stress signaling pathways, clarifying the drought resistance function of BnGS. After drought treatment for several days, Arabidopsis thaliana overexpressing BnGS showed stronger drought tolerance than the wild type. After drought stress, Arabidopsis thaliana overexpressing BnGS grew normally and significantly better than the wild type, with significantly increased drought tolerance. These results indicate that BnGS can participate in the molecular regulation of ramie drought tolerance adaptation, filling the research gap of GS genes in the field of drought and significantly enhancing their application value in drought-resistant breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The figure shows the KEGG pathway enrichment scatter plot of differentially expressed genes.

[0038] Figure 2 This is the electrophoresis diagram of the cloning results of BnGS, BnCLPB, and BnPCS1 genes, where M: DNA marker; 1: BnPCS1; 2: BnGS; 3: BnCLPB.

[0039] Figure 3 This is the phylogenetic tree analysis diagram of BnGS.

[0040] Figure 4 This is the phylogenetic tree analysis diagram of BnCLPB.

[0041] Figure 5 This is the phylogenetic tree analysis diagram of BnPCS1.

[0042] Figure 6 The bar graph shows the expression levels of BnGS, BnCLPB and BnPCS1 genes in ramie leaves after drought stress. Figure 6 A is BnGS; Figure 6 B is BnCLPB; Figure 6 C is BnPCS1.

[0043] Figure 7Figure 2 is the phenotypic characteristics of Arabidopsis plants overexpressing BnGS under drought stress. Figure 7 A is the PCR test of the T1 generation of transgenic Arabidopsis; Figure 7 B is the phenotype of overexpressed Arabidopsis under drought stress; Figure 7 C is the root length of overexpressing Arabidopsis under drought stress; Figure 7 D is the fresh weight of overexpressing Arabidopsis under drought stress.

[0044] Figure 8 Figure 2 is a phenotypic diagram of Arabidopsis plants overexpressing BnCLPB under drought stress, where: Figure 8 A is the phenotype of overexpressed Arabidopsis under drought stress; Figure 8 B is the root length of overexpressing Arabidopsis under drought stress; Figure 8 C is the fresh weight of overexpressing Arabidopsis under drought stress.

[0045] Figure 9 Figure 2 is a phenotypic diagram of Arabidopsis plants overexpressing BnPCS1 under drought stress, where: Figure 9 A is the phenotype of overexpressed Arabidopsis under drought stress; Figure 9 B is the root length of overexpressing Arabidopsis under drought stress; Figure 9 C is the fresh weight of overexpressing Arabidopsis under drought stress.

[0046] Figure 10 is a bar graph showing the osmotic substance content of Arabidopsis thaliana overexpressing BnGS under drought stress, Figure 10 A is the proline content; Figure 10 B is the sodium ion content; Figure 10 C is the potassium ion content.

[0047] Figure 11 is a bar graph showing the antioxidant and peroxide contents in Arabidopsis thaliana overexpressing BnGS under drought stress. Figure 11 A is superoxide dismutase (SOD) activity; Figure 11 B is the malondialdehyde content; Figure 11 C is the hydrogen peroxide content.

[0048] Figure 12 The bar graph shows the expression levels of related genes in Arabidopsis thaliana overexpressing BnGS under drought stress; Figure 12 A is AtNCED3, Figure 12 B is AtWRKY40, Figure 12 C is AtABI, Figure 12 D is AtCCS. DETAILED DESCRIPTION

[0049] The present invention is not limited to the following specific embodiments. Based on the disclosure of the present invention, a person skilled in the art may adopt a variety of other specific embodiments to implement the present invention. Any simple changes or modifications made to the design structure and concept of the present invention fall within the scope of protection of the present invention. It should be noted that the embodiments and features of the embodiments of the present invention may be combined with each other unless they conflict.

[0050] The present invention aims to provide a method for improving the drought resistance of plants by using the ramie glutathione synthetase gene BnGS. The method specifically includes the following steps:

[0051] (1) Screening of drought-resistant genes in ramie: We analyzed the transcriptome data of ramie under drought stress, screened out significantly enriched pathways, and selected the top-ranked glutathione metabolism pathway. Three differentially expressed genes with high expression levels were selected from this pathway: BnGS, BnCLPB, and BnPCS1, to explore their roles in drought response;

[0052] (2) Cloning and expression pattern analysis of ramie drought-tolerance genes: Three drought-tolerance-related genes (BnGS, BnCLPB, and BnPCS1) of ramie were cloned and bioinformatics analysis and expression pattern analysis of ramie leaves under drought stress were performed;

[0053] (3) Phenotypic identification of transgenic plants under drought stress: BnGS, BnCLPB, and BnPCS1 were introduced into Arabidopsis thaliana, and phenotypic analysis under drought stress revealed that only the BnGS-transgenic Arabidopsis thaliana was able to resist drought stress;

[0054] (4) Analysis of the drought resistance function of ramie BnGS: Further functional verification of BnGS was conducted. BnGS enhances the drought resistance of plants by regulating glutathione (GSH) metabolism, reactive oxygen species (ROS) scavenging and the synergistic mechanism of related transcription factors under drought stress.

[0055] In further detail, the method for improving the drought resistance of plants by using the ramie glutathione synthetase gene BnGS of the present invention comprises the following steps:

[0056] Step 1: Screening of genes related to ramie drought tolerance

[0057] The transcriptome data of ramie under drought stress were analyzed, and the significantly enriched pathways were screened, and the top-ranked glutathione metabolism pathway was selected ( Figure 1 ). Three differentially expressed genes with high expression levels were selected from this pathway: BnGS, BnCLPB, and BnPCS1 (Table 1);

[0058] Step 2: Cloning of drought-tolerance-related genes

[0059] 1. Design specific primers for BnGS, BnCLPB, and BnPCS1 genes using Primer 5.0 software;

[0060] 2. Extract total RNA from ramie leaves (RNA extraction kit provided by Vazyme);

[0061] 3. Reverse transcription synthesis of ramie cDNA;

[0062] 4. Using the reverse transcribed cDNA as a template, the cDNA sequences of the three genes were amplified by PCR;

[0063] 5. Separate PCR products by agarose gel electrophoresis;

[0064] 6. Purify the PCR product using the FastPure Gel DNA Extraction Mini Kit (Vazyme).

[0065] 7. Ligate the PCR product with the pMD18-T vector, and heat-shock transform the ligation product with SK2301 competent cells;

[0066] 8. Select positive clones and perform PCR using universal primers on the pMD18-T vector;

[0067] 9. After integrating the sequencing results using Editseq software, the full-length cDNA sequences of the three genes were obtained.

[0068] Step 3: Tissue expression analysis of genes

[0069] The leaves of the cutting seedlings of Ramie No. 1 were selected at 0, 6, 12, and 24 hours. The total RNA was extracted and reverse transcribed into cDNA as a template. Real-time fluorescence quantitative PCR was performed to detect the expression of genes. Ramie actin (β-actin) was used as an internal reference. The relative expression levels of genes were calculated using the SPSS software.

[0070] Step 4: Phenotypic identification of transgenic plants

[0071] BnGS, BnCLPB and BnPCS1 were respectively introduced into Arabidopsis thaliana. The resistance of these three genes to drought stress was analyzed through phenotypic analysis under drought stress. It was found that only BnGS could resist drought stress. Therefore, further functional verification of the BnGS gene was performed.

[0072] 1. Screening of positive transgenic Arabidopsis plants

[0073] The constructed pCAMBIA1300-35S-BnGS-myc, pCAMBIA1300-35S-BnCLPB-myc, and pCAMBIA1300-35S-BnPCS1-myc vectors were transformed into Agrobacterium tumefaciens GV3101 by heat shock and then transferred into wild-type Arabidopsis thaliana by inflorescence infection. Positive seedlings from the T0 generation were selected on 50 mg / L hygromycin MS plates. Selected plants were transplanted into nutrient soil and cultured for approximately three weeks in a 25°C incubator with a 16h / 8h photoperiod. DNA was extracted from leaves of the transgenic Arabidopsis thaliana and analyzed by PCR. Seeds from the harvested positive plants were screened and cultured to the T3 generation.

[0074] 2. Phenotypic Characterization of Transgenic Arabidopsis under Drought Stress

[0075] Wild-type and T3 Arabidopsis thaliana plants expressing three genes (two independent BnGS lines: BnGS-OE-2 and BnGS-OE-9; two independent BnCLPB lines: BnCLPB-OE-1 and BnCLPB-OE-3; and two independent BnPCS1 lines: BnPCS1-OE-3 and BnPCS1-OE-7) were seeded on 1 / 2 MS medium, placed at 4°C for purification for 2 days, and then transferred to a light-temperature incubator for incubation. Two weeks later, healthy Arabidopsis seedlings of uniform height and growth were selected and planted in polyethylene plastic pots with a perlite matrix. After 10 days of incubation, 20% PEG-200 was added to the Arabidopsis pots. No standard solution was added as a control. Each stress treatment was replicated three times. Seven days later, leaves were harvested, snap-frozen in liquid nitrogen, and stored at -80°C for measurement of physiological parameters and RNA extraction. Three biological replicates were used for each sample. The remaining culture medium on the washed roots was dried, and the root length and fresh weight were measured. The measurements were repeated three times.

[0076] Step 5: Analysis of the drought tolerance function of ramie BnGS

[0077] 1. Determination of Sodium and Potassium in Overexpressed Arabidopsis

[0078] After two weeks of abiotic treatment, the entire Arabidopsis plant was carefully removed, cleaned of dust and perlite with deionized water, dried with absorbent paper, and weighed and recorded. A portion was then sterilized at 105°C for 30 min and then dried at 65°C to constant weight. The sample was ground and sieved, and digested using the HNO₃-HClO₄ method. Heavy metal sodium and potassium contents were determined using a SOLAAR M6 atomic absorption spectrometer.

[0079] 2. Determination of Antioxidant Enzyme Activity and PCs Content in Overexpressed Arabidopsis

[0080] Superoxide dismutase (SOD), malondialdehyde (MDA) content, hydrogen peroxide (H2O2) content, and reduced ascorbic acid (AsA) and proline (Pro) activities were determined using kits from Shanghai Zhuocai Biotechnology Co., Ltd. All the above determinations were repeated three times.

[0081] 3. Expression of drought-tolerance-related genes

[0082] The expression of Arabidopsis thaliana 9-cis-epoxycarotenoid dioxygenase 3 (AtNCED3), WRKY transcription factor (AtWRKY40), ABI protein (Abscisic acid Insensitive, AtABI), and copper chaperone for superoxide dismutase (AtCCS) genes in transgenic plants was detected by qPCR.

[0083] The experiment was conducted in the Cultivation Technology and Fiber Material Engineering Laboratory in the 12th Teaching Building of Hunan Agricultural University. The experimental materials were ramie from the Ramie Institute of Hunan Agricultural University ( Boehmeria nivea ) Common variety Zhongzhu No. 1.

[0084] For this experiment, uniformly growing ramie cuttings (approximately 20 cm) were selected, soil removed from their roots, and transplanted into plastic pots containing perlite. Each pot was watered weekly with 500 mL of 1 / 2 Hoagland nutrient solution. Three weeks later, the plants were treated with 200 mmol / L mannitol. A control without mannitol was used. Three replicate pots were used for each treatment, with one plant per pot. Leaves were harvested at 0, 6, 12, and 24 hours after treatment, snap-frozen in liquid nitrogen, and stored at -80°C for analysis of gene expression in response to drought stress.

[0085] Arabidopsis thaliana Col-0, pCAMBIA1300-35S-BnGS-myc, pCAMBIA1300-35S-BnCLPB-myc, and pCAMBIA1300-35S-BnPCS1-myc overexpression vectors were prepared in previous experiments. The preparation method was based on the existing technology (Hu Min, Song Hui, et al. Construction and identification of Arabidopsis WRKY13 gene expression vector [J]. Journal of Hefei University of Technology (Natural Science Edition), 2024, 47(10): 1368-1371.).

[0086] Example 1

[0087] 1. Screening for genes related to drought tolerance in ramie

[0088] Ramie transcriptome analysis and screening of key genes: Analyze the ramie transcriptome data under drought stress, screen out significantly enriched pathways, and select the top-ranked glutathione metabolic pathways. Based on the ramie transcriptome data under drought stress, three glutathione metabolic pathways were screened ( Figure 1 Three differentially expressed genes with high expression levels were selected from this pathway: BnGS, BnCLPB, and BnPCS1 (Table 1).

[0089] Table 1 Screening of differentially expressed genes in the transcriptome

[0090]

[0091] From Table 1, it can be seen that the three genes were significantly differentially expressed in rhizomes.

[0092] 2. Cloning of drought-tolerance-related genes

[0093] Table 2 Primers used in the present invention

[0094]

[0095] Step 1: Gene cloning

[0096] 1. Specific primers for BnGS, BnCLPB, and BnPCS1 genes were designed using Primer 5.0 software, as shown in Table 2;

[0097] 2. Extract total RNA from ramie leaves (RNA extraction kit provided by Vazyme);

[0098] 3. Reverse transcription synthesis of ramie cDNA (the reverse transcription process was carried out according to the instructions of the reverse transcription kit R412, provided by Vazyme);

[0099] 4. Using the reverse-transcribed cDNA as a template, PCR amplification was performed to obtain cDNA sequences for the three genes. The reaction system consisted of 10 µL of 2× ChamQ Universal SYBR qPCR Master Mix, 0.4 µL of each upstream and downstream primer, 2 µL of cDNA, and 7.2 µL of ddH2O. The PCR protocol was as follows: 95°C for 30 s, 62°C for 30 s, and 72°C for 90 s for 33 cycles, followed by 72°C for 5 min.

[0100] 5. Separate PCR products by agarose gel electrophoresis: Prepare a 1% agarose gel (weigh 0.5 g agarose into a conical flask, add 50 ml 1× TAE buffer, and heat in a microwave oven until the agarose is completely dissolved. After the solution cools slightly, add nucleic acid dye and pour into a mold. After the gel solidifies, perform agarose gel electrophoresis. Load 10 μL of PCR product and DNA marker for electrophoresis at 120 V for 30 min.

[0101] 6. Purify the PCR product using the FastPure Gel DNA Extraction Mini Kit (Vazyme).

[0102] 7. The PCR product was ligated with the pMD18-T vector (the construction method of the pMD18-T vector refers to the existing technology (Wang Yun, Mei Miao, Yang Xue, Zhu Ling. Construction of a cloning vector recombinant plasmid pMD18-T Simple-PCV2 [J]. Heilongjiang Animal Husbandry and Veterinary Medicine, 2012, (11): 19-23)). The ligation process is as follows: first, take a sterile 1.5 mL EP tube and mark it. Then, add the pMD18-T vector, PCR product and Solution I in sequence according to the ligation reaction system. When adding, place the pipette tip against the tube wall to avoid generating bubbles. After adding, gently tap the bottom of the tube to mix the solution thoroughly. Incubate the ligation at 16 ℃ overnight. The ligation system (10 μL) is: pMD18-T Simple Vector 1 μL, PCR recovery product 4 μL, Solution I 5 μL. Obtain the ligation product. The ligation product was transformed with heat shock using SK2301 competent cells (the preparation method of competent cells can refer to the existing technology (Huang Xuejuan, Zhang Jindi, et al. An optimized preparation and transformation method of Escherichia coli competent cells [J]. Genomics and Applied Biology, 2017, 36 (12): 5199-5204.)) (the process of heat shock transformation can refer to the existing technology (Li Jianbei. Construction of marker-free transgenic rice using OsPDCD5 as a selection marker and co-transformation with the target gene [D]. Jiangxi Agricultural University, 2012)).

[0103] 8. Select positive clones and perform PCR with universal primers on the pMD18-T vector. Prepare a 25 μL reaction system in a 0.2 mL PCR tube on ice: add 2.5 μL 10× PCR Buffer (containing MgCl2, ... 2+), 2 μL dNTP Mix, 1 μL each of upstream and downstream primers, 1 μL template DNA, and finally 0.2 μL Taq DNA polymerase was added and filled with ddH2O to 25 μL. Gently centrifuge to mix and avoid bubbles. The reaction procedure is: 94 ℃ 5 min; 94 ℃ 30 s, 58 ℃ 30 s, 72 ℃ 1min / kb, (the time of the three genes is adjusted according to the length) 25 cycles; 72 ℃ 10 min; 4 ℃ storage. The PCR products were subjected to agarose gel electrophoresis using the same method as above. The results of agarose gel electrophoresis showed that the target fragments of the three genes were successfully amplified ( Figure 2 ).

[0104] 9. Purify the PCR product using the FastPure Gel DNA Extraction Mini Kit (Vazyme) and send it for sequencing (sequencing company: Hunan Qingke Biotechnology Co., Ltd. (Changsha)).

[0105] 9 After integrating the sequencing results using Editseq software, the full-length cDNA sequences of the three genes were obtained.

[0106]

[0107]

[0108] BnPCS1 sequence (SEQ ID NO. 23):

[0109]

[0110] Step 2: Bioinformatics analysis of the three genes BnGS, BnCLPB, and BnPCS1

[0111] 1. Based on the gene sequences of the three genes, use the Blast function of the NCBI website to perform online comparison to find the conserved domains of the three genes, and log in to the website (https: / / www.ncbi.nlm.nih.gov / orffinder / ) to find the open reading frames (ORFs) of the genes;

[0112] 2. Translate the BnGS nucleic acid sequence using the ExPASy online tool;

[0113] 3. Use ProtParam to analyze the physicochemical properties of the deduced protein;

[0114] 4. Use ProtScale to predict the hydrophilicity of proteins;

[0115] 5. Use wolf pSORT to predict subcellular localization;

[0116] 6. Use SOPMA online software to analyze the secondary structure of proteins;

[0117] 7. Use Phyre 2 online software to predict protein tertiary structure;

[0118] 8. Use DNAMAN 8 software to perform multiple alignment analysis on the protein sequences of ramie and other species.

[0119] The amino acid similarity search of BnGS, BnCLPB and BnPCS1 proteins was performed by NCBI BLASTp. The results showed that the ramie BnGS protein was similar to that of mustard ( Brassica juncea , O23732.1), tomato ( Solanum lycopersicum ,O22494.1), Arabidopsis thaliana ( Arabidopsis thaliana , P46416.3) have high amino acid sequence similarity, with similarities of 77.1%, 75.31% and 74.32% respectively. Multiple sequence alignment using DNAMAN revealed that BnGS is highly conserved among different species and has active sites involved in peptide ligation reactions in different species. The ramie BnCLPB protein is similar to the Arabidopsis thaliana CLpB3 ( Arabidopsis thaliana , Q9LF37.1), Arabidopsis CLpB4 ( Arabidopsis thaliana ,Q8VYJ7.1)、japonica rice CLpB2( Oryza sativa Japonica Group , Q75GT3.1), japonica rice CLpB3 ( Oryza sativa Japonica Group, Q0E3C8.3) have high amino acid sequence similarity, with similarities of 86.47%, 69.64%, 84.93% and 71.88% respectively. Multiple sequence alignment using DNAMAN showed that the CL37251 domain was highly conserved among different species; the ramie BnPCS1 protein was highly conserved with plum ( Prunus mume , XP_016650925.1) have high amino acid sequence similarity, with similarities of 78.56% and 78.56% respectively. Multiple sequence alignment using DNAMAN showed that the CL37251 domain was highly conserved among different species.

[0120] 9. Use MEGA11 software to complete the construction of phylogenetic tree, build phylogenetic tree based on amino acid sequences with high homology, and obtain the homology relationship between BnGS, BnCLPB, BnPCS1 and other plants through kinship. Figure 3 、 Figure 4 、 Figure 5 The results showed that the ramie BnGS protein was closely related to the mustard ( Brassica juncea , O23732.1), tomato ( Solanum lycopersicum , O22494.1), Arabidopsis thaliana ( Arabidopsis thaliana , P46416.3) and speculated that this type of protein has the same function in plants; the ramie BnCLPB gene is closely related to the Sichuan mulberry ( Morus notabilis , XM_024161122.1) and speculated that this type of gene may have similar functions; the ramie BnPCS1 gene and plum ( Prunus mume , XP_016650925.1), and it is speculated that this type of gene may have similar functions.

[0121] Step 3: Tissue expression analysis of genes

[0122] Two-week-old ramie seedlings were subjected to drought treatment. Leaves from the Zhongzhu No. 1 seedlings were sampled at 0, 6, 12, and 24 h post-treatment. Total RNA was extracted from the leaves (using the RNA extraction kit provided by Vazyme). The RNA was reverse-transcribed into cDNA, which served as a template (the reverse transcription process was performed according to the instructions of the reverse transcription kit R412, also provided by Vazyme). Real-time fluorescence quantitative PCR was used to amplify and detect gene expression. The reaction system consisted of 10 μL of 2× ChamQ Universal SYBR qPCR Master Mix, 0.4 μL of each 10 μmol / L upstream and downstream primers, 2 μL of cDNA, and 7.2 μL of ddH2O. Reaction conditions were: 95°C for 30 s; 95°C for 10 s, 60°C for 30 s, for 40 cycles. Ramie actin (β-actin) was used as an internal control. The relative expression levels of genes were calculated using the SPSS software.

[0123] Fluorescence quantitative analysis revealed that the transcriptional levels of the three genes were upregulated in leaves in response to drought stress ( Figure 6 The expression level of the BnGS gene reached its highest level after 12 hours of drought treatment, reaching 4.37 times that of the control group. The expression level of the BnCLPB gene reached its highest level after 6 hours of drought treatment, reaching 3.3 times that of the control group. The expression level of the BnPCS1 gene reached its highest level after 24 hours of drought treatment, reaching 2.24 times that of the control group. The BnGS, BnCLPB, and BnPCS1 genes were significantly upregulated at the transcriptional level in response to drought stress.

[0124] Step 4: Phenotypic identification of transgenic plants

[0125] BnGS, BnCLPB, and BnPCS1 were separately introduced into Arabidopsis thaliana. Phenotypic analysis of the drought resistance of these three genes revealed that only BnGS was able to resist drought stress. Therefore, BnGS was selected for further functional verification. The specific process is as follows:

[0126] Arabidopsis thaliana Col-0, pCAMBIA1300-35S-BnGS-myc, pCAMBIA1300-35S-BnCLPB-myc, and pCAMBIA1300-35S-BnPCS1-myc overexpression vectors were prepared in previous experiments. The preparation method was based on the existing technology (Hu Min, Song Hui, et al. Construction and identification of Arabidopsis WRKY13 gene expression vector [J]. Journal of Hefei University of Technology (Natural Science Edition), 2024, 47(10): 1368-1371.).

[0127] 1. Screening of positive transgenic Arabidopsis plants

[0128] The constructed pCAMBIA1300-35S-BnGS-myc, pCAMBIA1300-35S-BnCLPB-myc, and pCAMBIA1300-35S-BnPCS1-myc vectors were transformed into Agrobacterium tumefaciens GV3101 via heat shock (the heat shock transformation process is described in the prior art (Li Jianbei. Construction of Marker-Free Transgenic Rice Using OsPDCD5 as a Selectable Marker and Co-transformation with the Target Gene [D]. Jiangxi Agricultural University, 2012)) to obtain recombinant Agrobacterium. The vector construction process is described in the prior art (Jia Xingyue. Construction of the pCAMBIA1300-AFH16-GFP Recombinant Plasmid and Screening of Homozygous Transgenic Arabidopsis Plants [D]. Shanxi Normal University, 2017). The main process includes the following: 1. Target gene cloning: PCR amplify the BnGS gene and recover the purified fragment by gel; 2. Vector linearization: Use BamHI and KpnI to double-digest the pCAMBIA1300 vector, remove the original fragment, and recover the large fragment by gel; 3. Ligation and recombination: Mix the purified BnGS-myc PCR product and the linearized vector in a 3:1 molar ratio, use T4 DNA ligase to ligate overnight at 16°C; 4. Transform Escherichia coli, heat-shock the ligation product into DH5α competent cells, and coat kanamycin plates to screen positive clones; 5. Plasmid verification: Pick a single colony, extract the plasmid and perform enzyme digestion identification; 6: Sequencing verification to confirm the correctness of the gene sequence, reading frame and tag.

[0129] The recombinant Agrobacterium was then transferred into wild-type Arabidopsis using the inflorescence infection method (reference: Liu Huijuan, Feng Zhiguo, Li Xianwen, Li Tao, He Guangyuan. Obtaining transgenic Arabidopsis thaliana expressing crtB gene using the inflorescence infection method of Agrobacterium [J]. Hubei Agricultural Sciences, 2013, 52(01): 200-202). T0 generation seeds were planted on 50 mg / L hygromycin MS plates (culture medium formula: MS basic salt medium 4.4 g / L + sucrose 10 g / L + agar 8 g / L + 50 mg / L hygromycin). Seedlings that grew normally (root elongation and cotyledons turning green) on the MS plates were considered positive seedlings, and positive seedlings were screened. The selected plants were transplanted into nutrient soil and cultured for approximately 3 weeks in a 25°C incubator with a 16 h / 8 h photoperiod (16 h light, 8 h dark). RNA was extracted from transgenic Arabidopsis leaves, reverse transcribed, purified by PCR, and separated by gel electrophoresis. Sequencing and identification were performed (the same procedures as above). The PCR reaction system consisted of 1 μL DNA, 0.1 μL Taq enzyme, 1 μL 10× Buffer, 1 μL dNTPs, 1 μL each of the 2 μmol / L upstream and downstream primers (Table 2), and 4.9 μL ddH₂O. The PCR amplification protocol was as follows: initial denaturation at 97°C for 3 min; 30 cycles of denaturation at 95°C for 40 s, annealing at 58°C for 40 s, and extension at 72°C for 1 min; and a total extension at 72°C for 7 min. The seeds of the identified positive plants were further screened and cultured to the T3 generation of Arabidopsis (two independent strains of BnGS: BnGS-OE-2 and BnGS-OE-9; two independent strains of BnCLPB: BnCLPB-OE-1 and BnCLPB-OE-3; two independent strains of BnPCS1: BnPCS1-OE-3 and BnPCS1-OE-7) (the culture method was the same as before: seeds were planted on 50 mg / L hygromycin MS plates, and positive seedlings were selected. The screened plants were transplanted into nutrient soil and cultured in a 25°C light and temperature incubator with a 16 h / 8 h photoperiod (16 h light, 8 h dark) for about 3 weeks. DNA was extracted from the leaves of the transgenic Arabidopsis for PCR identification).

[0130] 2. Growth of transgenic Arabidopsis plants under drought stress

[0131] Wild-type and T3 Arabidopsis thaliana plants expressing three genes (two independent BnGS lines: BnGS-OE-2 and BnGS-OE-9; two independent BnCLPB lines: BnCLPB-OE-1 and BnCLPB-OE-3; and two independent BnPCS1 lines: BnPCS1-OE-3 and BnPCS1-OE-7) were seeded on 1 / 2 MS medium, placed at 4°C for purification for 2 days, and then transferred to a light-temperature incubator for incubation. Two weeks later, healthy Arabidopsis seedlings of uniform height and growth were selected and planted in polyethylene plastic pots with a perlite matrix. After 10 days of incubation, 20% PEG-200 was added to the Arabidopsis pots. No standard solution was added as a control. Each stress treatment was replicated three times. Seven days later, leaves were harvested, snap-frozen in liquid nitrogen, and stored at -80°C for measurement of physiological parameters and RNA extraction. Three biological replicates were used for each sample. The remaining culture medium on the washed roots was dried and the root length and fresh weight were measured. The measurements were repeated 3 times. Figure 7 、 Figure 8 、 Figure 9 shown.

[0132] RNA was extracted from transgenic Arabidopsis leaves, and reverse transcription and PCR were performed (the process was the same as above, and the primers were in Table 2). The 885 bp target band was successfully amplified from all 12 positive seedlings, indicating that BnGS, BnCLPB, and BnPCS1 were successfully introduced into Arabidopsis ( Figure 7 A). Compared with the control group, 20% PEG200 drought stress significantly inhibited the growth of BnCLPB and BnPCS1 Arabidopsis ( Figure 8 A, Figure 9 A), while the growth of BnGS Arabidopsis was not significantly inhibited ( Figure 7 B) Under drought conditions, the root length and fresh weight of the transgenic lines BnCLPB-OE-1, BnCLPB-OE-3, BnPCS1-OE-3, and BnPCS1-OE-7 were reduced compared to the wild type ( Figure 8 B. Figure 8 C and Figure 9 B. Figure 9 C), while the root length of BnGS-OE-2 and BnGS-OE-9 transgenic lines increased by 19.42% and 24.46%, and the fresh weight increased by 49.39% and 60.41%, respectively ( Figure 7 C. Figure 7 D) The results showed that overexpression of the BnGS gene significantly improved the drought tolerance of Arabidopsis thaliana.

[0133] 2. Determination of Sodium and Potassium in Arabidopsis Overexpressing BnGS

[0134] (1) Wild-type and T3 generation Arabidopsis thaliana (two independent lines, BnGS-OE-2 and BnGS-OE-9) were planted in 1 / 2 MS medium, placed at 4°C for purification for 2 days, and then moved to a light-temperature incubator for culture;

[0135] (2) After 2 weeks, healthy Arabidopsis seedlings of uniform height and growth were selected and planted in polyethylene plastic pots with perlite matrix and cultured for 10 days;

[0136] (3) After 10 days, 20% PEG200 was added to the Arabidopsis potted plants, with no standard solution added as the control. Each stress was repeated three times;

[0137] (4) After 2 weeks of treatment, carefully remove the whole Arabidopsis plant, wash away dust and perlite with deionized water, wipe dry with absorbent paper, and weigh and record.

[0138] (5) One portion was fixed at 105 °C for 30 min, then dried at 65 °C to constant weight, ground, sieved, and digested using the HNO3-HClO4 method;

[0139] (6) The sodium and potassium contents were determined using a SOLAAR M6 atomic absorption spectrometer, and the proline content was determined using a kit from Shanghai Zhuocai Biotechnology Co., Ltd. The results are as follows: Figure 10 As shown. Figure 10 It can be seen that after 2 weeks of drought treatment with 20% PEG200, the Pro content of BnGS-OE-2 and BnGS-OE-9 lines decreased by 46.08% and 53.37%, respectively ( Figure 10 A); the sodium ion content is 1.50 times and 1.24 times that of the wild type ( Figure 10 B); Potassium ion content is 1.47 times and 1.67 times that of the wild type ( Figure 10 C). This indicates that the BnGS gene regulates the absorption of Pro, sodium, and potassium, and maintains ion balance in Arabidopsis.

[0140] 3. Determination of Antioxidant Enzyme Activity and PCs Content in Arabidopsis Overexpressing BnGS

[0141] (1) Wild-type and T3 generation Arabidopsis thaliana (two independent lines, BnGS-OE-2 and BnGS-OE-9) were planted in 1 / 2 MS medium, placed at 4°C for purification for 2 days, and then moved to a light-temperature incubator for culture;

[0142] (2) After 2 weeks, healthy Arabidopsis seedlings of uniform height and growth were selected and planted in polyethylene plastic pots with perlite matrix and cultured for 10 days;

[0143] (3) After 10 days, 20% PEG200 was added to the Arabidopsis potted plants, with no standard solution added as the control. Each stress was repeated three times;

[0144] (4) After 2 weeks of treatment, carefully remove the whole Arabidopsis plant, wash away dust and perlite with deionized water, wipe dry with absorbent paper, and weigh and record.

[0145] (5) One portion was fixed at 105 °C for 30 min, then dried at 65 °C to constant weight, ground, sieved, and digested using the HNO3-HClO4 method;

[0146] (6) Superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, and hydrogen peroxide (H2O2) content were determined using kits from Shanghai Zhuocai Biotechnology Co., Ltd. The above determinations were repeated three times. Figure 11 As shown. 20% PEG200 drought stress increased the SOD of Arabidopsis thaliana ( Figure 11 A), MDA ( Figure 11 B) and H2O2 ( Figure 11 C). Arabidopsis overexpressing BnGS exhibited significantly improved antioxidant capacity compared to the wild type (P < 0.05). Compared to the wild type, the superoxide dismutase (SOD) activity of BnGS-OE-2 and BnGS-OE-9 increased by 67.90% and 77.85%, respectively. MDA content decreased by 24.68% and 36.00%, respectively, and H₂O₂ content decreased by 33.49% and 18.33%, respectively, in BnGS-OE-2 and BnGS-OE-9. This suggests that BnGS overexpression in Arabidopsis mitigates oxidative damage under drought stress by enhancing the antioxidant defense system.

[0147] 4. Detection of the expression of abiotic stress genes in transgenic plants overexpressing BnGS Arabidopsis

[0148] (1) Extraction of Arabidopsis thaliana total RNA was performed using the FastPure Universal Plant Total RNA Isolation Kit (Novagene Biotech Co., Ltd., Nanjing).

[0149] (2) The integrity and purity of the obtained RNA were detected by 1.5% agarose gel electrophoresis and UV spectrophotometry;

[0150] (3) PrimerPremier 5.0 software was used to design real-time fluorescence quantitative PCR primers (Table 2) and synthesized by Hunan Qingke Biological Co., Ltd. (Changsha);

[0151] (4) The expression of Arabidopsis thaliana 9-cis-epoxycarotenoid dioxygenase 3 (AtNCED3), WRKY transcription factor (AtWRKY40), Abscisic acid insensitive (AtABI), and copper chaperone for superoxide dismutase (AtCCS) genes in transgenic plants was detected using qPCR. The reaction system included 10 μL of 2×ChamQ Universal SYBR qPCR Master Mix, 0.4 μL of 10 μmol / L upstream and downstream primers, 2 μL of cDNA, and 7.2 μL of ddH2O. The reaction conditions were: 95°C for 30 s; 95°C for 10 s, 60°C for 30 s, and 40 cycles. Each sample was repeated three times.

[0152] (5) Using Arabidopsis β-actin8 as the internal reference gene, The relative expression of genes was calculated by

[0153] (6) SPSS 7.0 and Microsoft Excel 2016 were used to perform statistical analysis of the data. Figure 12 As shown, Figure 12 A is AtNCED3, Figure 12 B is AtWRKY40, Figure 12 C is AtABI, Figure 12 D is AtCCS. The expression levels of these four genes in Arabidopsis plants overexpressing BnGS after drought stress were higher than those in wild-type plants. Under drought stress, the expression levels of AtNCED3 were 4.80 and 4.47 times that of the wild-type, AtWRKY40 were 3.22 and 4.01 times that of the wild-type, AtABI were 3.68 and 5.78 times that of the wild-type, and AtCCS were 2.71 and 1.62 times that of the wild-type. Figure 12 ). These results indicate that overexpression of the BnGS gene can effectively regulate the transcriptional levels of multiple key stress-responsive genes, including AtNCED3, AtWRKY40, AtABI, and AtCCS, in Arabidopsis thaliana under drought stress conditions.

[0154] Conclusion: In this study, under drought stress, Arabidopsis plants overexpressing BnGS showed higher root length, biomass, and sodium and potassium contents than the wild type. They also exhibited significant advantages in antioxidant enzyme activity, while MDA and H₂O₂ contents were significantly lower than those of the wild type. This suggests that overexpressing BnGS in Arabidopsis can enhance antioxidant capacity, eliminate excess free radicals, and improve plant drought resistance.

[0155] It should be noted that the above embodiments are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. A person skilled in the art would be able to make other variations or modifications based on the above description. It is not possible to enumerate all embodiments here. Any obvious variations or modifications arising from the technical solution of the present invention remain within the scope of protection of the present invention.

Claims

1. Application of ramie glutathione synthetase gene in regulating plant drought resistance, characterized in that: The sequence of the ramie glutathione synthetase gene is shown in SEQ ID NO. 1; and the plant is Arabidopsis thaliana.

2. The use according to claim 1, characterized in that The application is as follows: cloning the ramie glutathione synthetase gene and constructing an overexpression vector, and then transfecting the overexpression vector into wild-type plants to obtain ramie glutathione synthetase gene overexpressing plants, which are drought-resistant plants.

3. Use of a reagent for increasing the expression of ramie glutathione synthetase gene in the preparation of a reagent for regulating plant drought resistance, characterized in that: The reagent for increasing the expression of ramie glutathione synthetase gene includes one or more of the following: A, primers for amplifying ramie glutathione synthetase gene; B. a kit containing primers for amplifying the ramie glutathione synthetase gene; C. a recombinant vector containing primers for amplifying the ramie glutathione synthetase gene; D, Agrobacterium containing primers for amplifying the ramie glutathione synthetase gene; The plant is Arabidopsis thaliana.

4. The use according to claim 3, characterized in that The recombinant vector containing the primers for amplifying the ramie glutathione synthetase gene is pCAMBIA1300-35S-BnGS-myc.

5. The use according to claim 3, characterized in that The sequences of primers for amplifying the ramie glutathione synthetase gene are shown in SEQ ID NO.4 and SEQ ID NO.

5.

6. Use of a reagent for increasing the expression of ramie glutathione synthetase gene in the preparation of a reagent for regulating plant growth ability, characterized in that: The reagent for increasing the expression of ramie glutathione synthetase gene includes one or more of the following: A, primers for amplifying ramie glutathione synthetase gene; B. a kit containing primers for amplifying the ramie glutathione synthetase gene; C. a recombinant vector containing primers for amplifying the ramie glutathione synthetase gene; D. Agrobacterium containing the recombinant vector; The plant is Arabidopsis thaliana; The plant growth capacity includes root length, biomass, sodium content and potassium content.

7. Use of a reagent for increasing the expression of ramie glutathione synthetase gene in the preparation of a reagent for regulating plant antioxidant activity, characterized in that: The reagent for increasing the expression of ramie glutathione synthetase gene includes one or more of the following: A, primers for amplifying ramie glutathione synthetase gene; B. a kit containing primers for amplifying the ramie glutathione synthetase gene; C. a recombinant vector containing primers for amplifying the ramie glutathione synthetase gene; D, Agrobacterium containing primers for amplifying the ramie glutathione synthetase gene; The plant is Arabidopsis thaliana.

8. The use according to any one of claims 3, 6 or 7, characterized in that The regulation is increasing.

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