Application of GhGRD1 and / or GhGRD2 genes in enhancing plant drought resistance

Abstract

The present invention discloses GhGRD1 and / or GhGRD2 The present invention belongs to the field of plant genetic engineering technology. GhGRD1 and GhGRD2 The gene was isolated and cloned from the homologous gene on the AD genome of upland cotton. GhGRD1 / 2 The recombinant vector of the gene was transferred into cotton plants and silenced after drought stress treatment. GhGRD1 / 2 Cotton plants expressing the gene are drought sensitive, and the overexpression GhGRD1 and GhGRD2 The recombinant vector of the gene was transferred into Arabidopsis plants and overexpressed GhGRD1 and GhGRD2 Arabidopsis plants with this gene are more drought-resistant, providing genetic resources for molecular breeding of plant drought resistance.

Description

Technical Field

[0001] The present invention belongs to the field of plant genetic engineering technology and specifically relates to GhGRD1 and / or GhGRD2 Application of genes in enhancing drought resistance in plants. Background Art

[0002] Drought refers to a shortage of available water, resulting in reduced yields, or a period of no rainfall or irrigation, which impacts crop growth. When plants are subjected to drought stress, reactive oxygen species (ROS) accumulate in their bodies. While a moderate amount of ROS can help plants withstand adverse conditions, an excessive amount can cause damage. When plants are subjected to drought stress, the burst of ROS leads to the accumulation of related compounds such as hydrogen peroxide (H2O2) and malondialdehyde (MDA), generating lipid-derived free radicals that exacerbate oxidative reactions and damage plant cell membranes. To eliminate excess ROS, plants have evolved complex enzymatic and non-enzymatic systems to eliminate the oxidative damage caused by ROS and maintain a dynamic balance of ROS.

[0003] Xinjiang, my country's largest cotton-producing region, is located in arid and semi-arid areas. Drought stress affects cotton growth, yield, and quality. Because cotton grows under long-term drought stress, it easily accumulates large amounts of reactive oxygen species during growth. Therefore, research on enzyme genes that scavenge reactive oxygen species and the use of genetic engineering to cultivate drought-tolerant varieties effectively mitigate the impact of drought stress on plant growth and development are of great research value. Summary of the Invention

[0004] The object of the present invention is to provide GhGRD1 and / or GhGRD2 Application of genes in enhancing drought resistance in plants.

[0005] In order to achieve the above object, the technical solution of the present invention is as follows:

[0006] Upland cotton GhGRD1 Genes and GhGRD2 The gene is a homologous gene on the upland cotton AD genome, GhGRD1 The CDS sequence of the gene is shown in SEQ ID NO.1. GhGRD2 The CDS sequence of the gene is shown in SEQ ID NO.2. GhGRD1 The amino acid sequence of the GhGRD1 protein encoded by the gene is shown in SEQ ID NO.3. GhGRD2 The amino acid sequence of the GhGRD2 protein encoded by the gene is shown in SEQ ID NO.4.

[0007] The present invention also constructs a series of plant expression vectors, and the expression vectors, recombinant vectors or transgenic plant lines containing the above genes and the host cells containing the vectors also fall within the scope of protection of the present invention in terms of enhancing plant drought resistance. GhGRD1 and GhGRD2 Gene overexpression or overexpression recombinant vector is used to obtain cotton or Arabidopsis plants with improved drought resistance.

[0008] The functions of the genes protected by the present invention include not only the above GhGRD1 and GhGRD2 Genes, including GhGRD1 and GhGRD2 The homologous genes with high homology (up to 99%) have functions in plant drought stress resistance.

[0009] The present invention discloses GhGRD1 and GhGRD2 The biological function of genes in plant drought resistance is specifically manifested in: under drought stress, GhGRD1 and GhGRD2 The degree of leaf wilting and yellowing in the gene silenced lines was higher than that in the control. GhGRD1 and GhGRD2 The degree of leaf yellowing in the overexpressing Arabidopsis lines was lower than that in the wild type.

[0010] According to its function, plants with improved drought resistance can be obtained by genetic modification. Specifically, GhGRD1 and GhGRD2 The gene is introduced into the target plant to obtain a transgenic plant, which has higher drought resistance than the target plant.

[0011] Specifically, GhGRD1 and GhGRD2 The gene can be introduced into the target plant via the recombinant expression vector. In the method, the recombinant expression vector can be used to transform plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, and Agrobacterium-mediated transformation, and the transformed plant tissues can be cultivated into plants.

[0012] The present invention also protects a method for regulating plant drought resistance, which is the following (1) or (2) or (3):

[0013] (1) By increasing the activity of GhGRD1 and GhGRD2 proteins in target plants, plants with stronger drought resistance than target plants can be obtained;

[0014] (2) By promoting the growth of target plants GhGRD1 and GhGRD2 Gene expression can produce plants with stronger drought resistance than the target plant;

[0015] (3) By inhibiting the GhGRD1 and GhGRD2 By regulating the expression of the gene, plants with lower drought resistance than the target plant are obtained.

[0016] “Promote the growth of target plants GhGRD1 and GhGRD2 The implementation of "gene expression" can be as follows (1) or (2) or (3):

[0017] (1) GhGRD1 and GhGRD2 Gene introduction into target plants;

[0018] (2) introduction of strong promoters or enhancers;

[0019] (3) Other common methods in this field.

[0020] Preferably, you can choose GhGRD1 and GhGRD2 Gene overexpression or overexpression.

[0021] “Inhibit the GhGRD1 and GhGRD2 Gene expression" by silencing or targeted mutagenesis GhGRD1 and GhGRD2 Gene.

[0022] In the present invention, there is no particular limitation on the plants suitable for the present invention, as long as they are suitable for gene transformation, such as various crops, flower plants, or forestry plants. The plants can be, for example (but not limited to): dicots, monocots, or gymnosperms.

[0023] As a preferred embodiment, the "plant" includes but is not limited to: cotton, Arabidopsis thaliana, especially upland cotton ( Gossypiumbirsutum ), all genes that have this gene or are homologous to it are applicable.

[0024] As used herein, "plant" includes the entire plant, its parent and progeny plants, and various parts of the plant, including seeds, fruits, buds, stems, leaves, roots (including tubers), flowers, tissues, and organs, all of which contain the gene or nucleic acid of interest. "Plant" as used herein also includes plant cells, suspension cultures, callus, embryos, meristematic regions, gametophytes, sporophytes, pollen, and microspores, each of which may contain the gene / nucleic acid of interest.

[0025] The present invention encompasses any plant cell, or any plant obtained or obtainable by any of the methods herein, and all plant parts and propagules thereof. The present invention also encompasses transfected cells, tissues, organs, or whole plants obtained by any of the aforementioned methods. The only requirement is that the progeny exhibit the same genotypic or phenotypic characteristics, and that the progeny obtained using the methods of the present invention have the same characteristics.

[0026] The present invention also extends to the harvestable parts of the plants as described above, but is not limited to seeds, leaves, fruits, scapes, roots, rhizomes, tubers and bulbs, and further to other derivatives of the plants after harvest, such as dry granules or powders, oils, fats and fatty acids, starch or proteins.

[0027] Advantages of the present invention:

[0028] (1) The present invention adopts the comparative transcriptomics method to innovatively analyze the upland cotton ( Gossypiumhirsutum ) that respond to adverse stress and participate in antioxidant regulation GhGRD1 and GhGRD2 (collectively known as GhGRD1 / 2 ) was cloned. The prokaryotic expression vector 6P1-GhGRD1 / 2 was constructed and the recombinant vector was transformed into Escherichia coli expression competent cells Rosette (DE3). GST-GhGRD1 / 2 was induced to express in large quantities and purified. The results showed that GhGRD1 / 2 had peroxidase activity and redox state. Further construction GhGRD1 / 2 Gene silencing vector TRV2-GhGRD1 / 2 was used to inject Agrobacterium containing TRV2-GhGRD1 / 2 into cotton leaves to obtain GhGRD1 / 2 The results showed that the leaves of the gene-silenced plants were severely wilted and yellowed under drought stress, indicating that they were more sensitive to drought treatment. GhGRD1, GhGRD2 The overexpression vectors p35S-GhGRD1-GFP and p35S-GhGRD2-GFP were used to transform wild-type Arabidopsis thaliana (C1o-0, WT) using the Agrobacterium inflorescence infection method. The overexpression plants were obtained. The analysis results showed that under drought stress, compared with the wild type, GhGRD1 as well as GhGRD2 The overexpression lines showed an increase in fresh weight and a significant decrease in H2O2 content, indicating that GhGRD1 as well as GhGRD2 The overexpression lines are more drought-resistant, providing genetic resources for molecular breeding of drought-resistant crops.

[0029] (2) In the actual breeding process, drought-resistant plants can be obtained by genetic modification. Specifically, GhGRD1 / 2The gene is introduced into the target plant to obtain a transgenic plant, which has higher drought resistance than the target plant, providing a new approach for plant drought resistance breeding.

[0030] (3) This invention discloses for the first time that GhGRD1 can interact with GhPSBO2 and GhIBI1 in plants, providing a basis for in-depth research on drought resistance gene pathways. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 yes GhGRD1 / 2 Analysis of gene expression in different tissues of upland cotton.

[0032] Figure 2 is the subcellular localization analysis of GhGRD1 and GhGRD2.

[0033] Figure 3 It's Gh GRD1 / 2 Gene silenced plants ( TRV:GhGRD1 / 2 ) and control plants ( TRV:00 ) Analysis of water loss rate and pore diameter; in the figure, A is the comparison of water loss rate; B and C are the comparison of pore diameter.

[0034] Figure 4 Gh under drought stress GRD1 / 2 Gene silenced plants ( TRV:GhGRD1 / 2 ) and control plants ( TRV:00 ) phenotypic analysis; in the figure, A is the phenotypic comparison; B, C, D, and E are the comparisons of plant height, root-to-shoot ratio, fresh weight, and survival rate, respectively.

[0035] Figure 5 It is silent Gh GRD1 / 2 Genetic plants ( TRV:GhGRD1 / 2 ) and control plants ( TRV:00 ) on the antioxidant capacity of cotton; in the figure, A is the comparison of hydrogen peroxide content; B is the comparison of superoxide anion content; C is the comparison of leaf phenotype; D is the comparison of GPX activity; E is the comparison of malondialdehyde content; F is the comparison of ion leakage rate.

[0036] Figure 6 Overexpression of Gh GRD1 Phenotypic analysis of Arabidopsis plants and the control (WT); in the figure, A is the phenotypic comparison; B, C, D, and E are the comparisons of water loss rate, fresh weight, hydrogen peroxide content, and survival rate, respectively.

[0037] Figure 7 Overexpression of Gh GRD2 Phenotypic analysis of Arabidopsis plants and the control (WT); in the figure, A is the phenotypic comparison; B, C, D, and E are the comparisons of water loss rate, fresh weight, hydrogen peroxide content, and survival rate, respectively.

[0038] Figure 8 GhGRD1 yeast autoactivation and toxicity assays.

[0039] Figure 9 This is the screening and verification of GhGRD1 interacting proteins; in the figure, A is the interaction between GhGRD1 and GhGIS and GhMIEL1 respectively; B is the interaction between GhGRD1 and GhPSBO2 and GhIBI1 respectively.

[0040] Figure 10 Subcellular localization analysis of GhPSBO2 and GhIBI1.

[0041] Figure 11 Verification of the in vivo interaction between GhGRD1, GhPSBO2 and GhIBI1. DETAILED DESCRIPTION

[0042] The present invention will be further described below with reference to specific examples, and the advantages and features of the present invention will become more apparent as the description proceeds. However, the specific experimental methods involved in the following examples, unless otherwise specified, are all conventional methods or are performed under the conditions recommended by the manufacturer's instructions.

[0043] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The experimental methods in the following examples are all conventional methods unless otherwise specified. Unless otherwise specified, the reagents and materials used can be purchased from the market.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0045] The present application will be further explained below in conjunction with the examples. Before introducing the specific examples, some of the biological materials, experimental reagents and other issues in the following examples are briefly explained as follows.

[0046] Biomaterials

[0047] Cotton TM-1 seeds were stored in the laboratory; Arabidopsis Col-0 seeds were stored in the laboratory;

[0048] Prokaryotic expression vector pGEX6P1; Gene silencing vector empty vector and positive control vector TRV::GhCLA Preserved in the laboratory; overexpression vector pSuper-1300-GFP Save for the laboratory;

[0049] Escherichia coli DH5α and Agrobacterium GV3101 Save for the laboratory;

[0050] Primer synthesis and sequencing were completed by Zhengzhou Qingke Biological Company.

[0051] Experimental reagents

[0052] RNA extraction kit, reverse transcription kit, and fluorescence quantification kit were purchased from Novozyme Biotechnology Co., Ltd.;

[0053] Common reagents such as NaCl were purchased from Solebol;

[0054] Hygromycin was purchased from Solebio Biotechnology;

[0055] MS culture medium was purchased from Beijing Coolaibo Technology Co., Ltd.;

[0056] Various endonucleases were purchased from Mona Biotechnology Co., Ltd.;

[0057] One-step cloning enzyme was purchased from Novozyme Biotechnology Co., Ltd.;

[0058] Plasmid miniprep kit and gel recovery kit were purchased from Beijing Tiangen Biotechnology Co., Ltd.

[0059] Experimental equipment

[0060] The PCR instrument was purchased from Bio-rad;

[0061] The refrigerated centrifuge was purchased from Eppendorf;

[0062] The quantitative PCR instrument was purchased from Bio-rad;

[0063] The laser confocal microscope was purchased from Zeiss;

[0064] The high-temperature and high-pressure sterilizer MLS-3750 was purchased from Sanyo Company, Japan;

[0065] The nucleic acid detector Nanodrop 2000C was purchased from ThermoScientific;

[0066] The room temperature centrifuge and microplate reader SpectraMax iD5 were purchased from Thermo Scientific.

[0067] The present invention screened two genes related to cotton drought resistance through biological technology: GhGRD1, GhGRD2 The functions of the genes were then verified by gene silencing and overexpression, as follows.

[0068] Example 1 GhGRD1, GhGRD2 Gene cloning

[0069] DNA of upland cotton was extracted, and the gene sequences obtained from the database CottonMD (https: / / yanglab.hzau.edu.cn / CottonMD) were cloned by PCR reaction using specific primers designed by Primer Premier. GhGRD1, GhGRD2 The CDS sequences of the genes are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively. GhGRD1, GhGRD2 The protein sequences corresponding to the CDS sequences of the genes are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively.

[0070] Example 2 GhGRD1 / 2 Gene expression analysis and fluorescence localization

[0071] To explore GhGRD1 / 2 Gene (i.e. GhGRD1 and GhGRD2 To investigate the expression patterns of the gene (same below) in different tissues of upland cotton, we collected tissue samples such as seeds, roots, stems, leaves, stigmas, ovules, petals, and anthers of upland cotton, extracted RNA, and reverse transcribed it into cDNA. We then used qPCR technology to detect GhGRD1 / 2 The relative expression levels of genes in various tissues. The results showed that the expression level in seeds was used as a reference. GhGRD1 / 2 The gene is expressed in various tissues, with higher expression levels in leaves, stigmas, petals, and anthers ( Figure 1 This result shows that GhGRD1 / 2 It is expressed in all tissues of cotton and may play a role in different stages of cotton growth and development.

[0072] To further analyze the characteristics of GhGRD1 and GhGRD2 proteins, we GhGRD1 and GhGRD2 The CDS sequences of the genes were cloned into the 35S::GFP vector, and the corresponding fusion protein expression vector was successfully constructed. The fusion protein was expressed in tobacco leaves using the Agrobacterium-mediated transient transformation method, and the transformed tobacco leaves were observed under a Nikon laser confocal microscope. The results showed that the GFP protein in the empty vector showed normal fluorescence signals in the cell nucleus and cytoplasm. The green fluorescence signals of GhGRD1:GFP and GhGRD2:GFP proteins overlapped and co-localized with the red fluorescence signals of H2B:RFP (nuclear localization protein), and the green fluorescence signals of GhGRD1:GFP and GhGRD2:GFP were also observed in the cytoplasm, indicating that GhGRD1 and GhGRD2 proteins were localized in the cell nucleus and cytoplasm ( Figure 2This result suggests that GhGRD1 and GhGRD2 may exert specific functions through the nucleus and cytoplasm.

[0073] Example 3 Silence GhGRD1 / 2 Genetic verification of its impact on cotton drought resistance

[0074] For preliminary exploration GhGRD1 / 2 To investigate the response of gene silencing to drought stress in cotton, we measured water loss and stomatal aperture in control plants (TRV:00) and silenced plants (TRV:GhGRD1 / 2) three weeks after Agrobacterium injection. The second true leaf of the cotton plant was placed on a balance on a water loss platform and its water loss rate was measured. The results showed that the water loss rate of the silenced plant was significantly increased compared to the control plant ( Figure 3 At the same time, the lower epidermis of the second true leaf of the control plant and the silenced plant was torn off with tweezers. The stomatal aperture of the cotton was observed under a microscope and the stomatal aperture was statistically analyzed. The results showed that the stomatal aperture of the silenced plant was significantly larger than that of the control plant ( Figure 3 The results showed that silencing the GhGRD1 / 2 gene made cotton more sensitive to drought stress.

[0075] To investigate the function of GhGRD1 / 2 genes in cotton drought response, we subjected control plants (TRV:00) and silenced plants (TRV: GhGRD1 / 2) three weeks after Agrobacterium injection to natural drought treatment. After 20 days of drought treatment, it was observed that under normal conditions (Mock), there was no significant difference in the growth status of the control plants and silenced plants. However, under drought conditions (Drought), compared with the control plants, the silenced plants showed a more wilted leaf phenotype and a worse growth state. After 3 days of re-watering, the control plants basically recovered to a near-normal growth state, while the silenced plants still showed a growth state with wilted leaves ( Figure 4 In addition, we analyzed the plant height, fresh weight, and root-to-shoot ratio of the control and silenced plants under drought stress, as well as the survival rate of the cotton after rehydration. The results showed that under normal conditions, there was no significant difference in plant height, root-to-shoot ratio, and fresh weight between the two plants; however, under drought conditions, the plant height and root-to-shoot ratio of the silenced plants were significantly reduced compared to the control plants ( Figure 4 B and C in Figure ), the fresh weight of the silenced plants also showed a downward trend ( Figure 4 D in the figure), and after rehydration, the survival rate of the control plants was significantly higher than that of the silent plants ( Figure 4 These results indicate that silencing the GhGRD1 / 2 gene reduces cotton tolerance to drought stress.

[0076] To investigate whether the GhGRD1 / 2 gene responds to drought stress by scavenging ROS, we measured ROS-related physiological indicators in control plants (TRV:00) and silenced plants (TRV:GhGRD1 / 2). The results showed that under normal growth conditions, there were no significant differences in hydrogen peroxide (H2O2), superoxide anion (O2⁻), malondialdehyde (MDA) content, glutathione peroxidase (GPX) activity, and ion leakage rate between the control and silenced plants. There was also no significant difference in the results of diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining ( Figure 5 ); However, under drought conditions, we found that the H2O2 and O2⁻ contents in gene-silenced plants were significantly higher than those in control plants ( Figure 5 A and B in the figure). After DAB and NBT staining, the leaves of the silenced plants were darker than those of the control plants ( Figure 5 C in the figure) showed that the leaves of the silenced plants accumulated more ROS under drought stress, and the GPX activity of the silenced plants was significantly lower than that of the control plants ( Figure 5 D in the figure further indicates that silencing the GhGRD1 / 2 gene leads to a decrease in the ability of cotton leaves to scavenge ROS under drought stress. In addition, under drought treatment conditions, the MDA content and ion leakage rate of silenced plants were significantly higher than those of control plants ( Figure 5 Figures E and F show that silencing GhGRD1 / 2 leads to more severe oxidative damage in cotton leaves under drought stress. These results indicate that silencing the GhGRD1 / 2 gene leads to increased ROS accumulation in cotton leaves and reduced antioxidant capacity, confirming that GhGRD1 / 2 can respond to cotton drought stress by scavenging ROS.

[0077] Example 4 Overexpression in Arabidopsis GhGRD1 and GhGRD2 Genetic verification of its impact on drought resistance

[0078] To further investigate the function of GhGRD1 / 2 genes in plant drought response, we constructed overexpression vectors 35S::GhGRD1:GFP and 35S::GhGRD2:GFP, respectively, and transformed them into Agrobacterium GV3101. We obtained Arabidopsis thaliana overexpressing GhGRD1 and GhGRD2 by inflorescence dip infection, and then screened positive transgenic plants using MS medium containing hygromycin to obtain pure lines.

[0079] To investigate the function of GhGRD1 in drought response in Arabidopsis, we measured the water loss rate of WT and GhGRD1-overexpressing Arabidopsis plants. The results showed that the water loss rate of the two GhGRD1-overexpressing Arabidopsis plants was significantly lower than that of the WT plants ( Figure 6A in the figure) indicates that overexpressing GhGRD1 Arabidopsis plants can improve the drought resistance of Arabidopsis. Next, we subjected WT plants and GhGRD1-overexpressing Arabidopsis plants to natural drought treatment. After 25 days of drought treatment, it was observed that under normal conditions (Mock), there was no significant difference in the growth status of WT plants and the two GhGRD1-overexpressing Arabidopsis plants, and they grew well; however, under drought conditions (Drought), the WT plant WT showed significant wilting and dehydration, and the growth condition of the two GhGRD1-overexpressing Arabidopsis plants was significantly better than that of the WT plant. After 3 days of rehydration (Re-watered), the two GhGRD1-overexpressing Arabidopsis plants basically recovered to a near-normal growth state, while the WT plant still showed a wilting and dehydration state ( Figure 6 In addition, we measured the fresh weight and H2O2 content of WT plants and two GhGRD1-overexpressing Arabidopsis plants under drought stress, as well as the survival rate of Arabidopsis plants after rehydration. The results showed that under normal conditions, there was no significant difference in fresh weight and H2O2 content between WT plants and the two GhGRD1-overexpressing Arabidopsis plants. However, under drought conditions, the fresh weight of the two GhGRD1-overexpressing Arabidopsis plants was significantly increased, and the H2O2 content was significantly decreased compared to WT plants ( Figure 6 C and D in Figure ), after rehydration, the survival rate of WT plants was significantly lower than that of the two Arabidopsis plants overexpressing GhGRD1 ( Figure 6 These results indicate that overexpression of the GhGRD1 gene improves the tolerance of Arabidopsis to drought stress, suggesting that GhGRD1 plays an important role in plant drought response.

[0080] The same method was used to analyze WT and overexpressed GhGRD2 The Arabidopsis plants were subjected to water loss rate measurement, natural drought treatment, fresh weight statistics, H2O2 content measurement and survival rate statistics. GhGRD2 The water loss rate of Arabidopsis plants was significantly lower than that of WT plants ( Figure 7 (A) After 25 days of drought treatment, it was observed that under normal conditions (Mock), the WT plants and the two overexpressing GhGRD2 There was no significant difference in the growth status of Arabidopsis plants; however, under drought conditions, the rosette leaves of WT plants showed a more wilted state, and the two overexpression GhGRD2 The growth of Arabidopsis plants was significantly better than that of WT plants. After re-watering for 3 days, the two overexpression GhGRD2 The Arabidopsis plants basically recovered to a near-normal growth state, while the WT plants still showed a growth state with wilting rosette leaves ( Figure 7In addition, we investigated the effects of drought stress on WT plants and two overexpressing GhGRD2 The fresh weight and H2O2 content of Arabidopsis plants were measured, and the survival rate of Arabidopsis plants after rehydration was calculated. The results showed that under normal conditions, the WT plants and the two overexpressing GhGRD2 There were no significant differences in fresh weight and H2O2 content of Arabidopsis plants; however, under drought conditions, the two lines overexpressing GhGRD2 The fresh weight of Arabidopsis plants increased significantly, and the H2O2 content decreased significantly ( Figure 7 C and D) After rehydration, the survival rate of WT plants was significantly lower than that of the two overexpressing GhGRD2 Arabidopsis plants ( Figure 7 This result indicates that overexpression GhGRD2 The gene improves the tolerance of Arabidopsis plants to drought stress, further confirming that GhGRD2 It also plays an important role in drought response.

[0081] Example 5 Study on the self-activation ability of GhGRD1 protein

[0082] To test whether the GhGRD1 protein is toxic to the yeast strain Y2H Gold and whether it can activate a reporter gene on its own, we constructed the GhGRD1 gene into the pGBKT7 vector and tested its self-activation and toxicity to eliminate potential effects of the protein itself on subsequent experiments. To ensure the reliability of the experimental results, we used plasmids pGBKT7:Lam and pGADT7:T as negative controls, and plasmids pGBKT7:53 and pGADT7:T as positive controls. Subsequently, three vectors (pGBKT7:GhGRD1 + pGADT7, pGBKT7:53 + pGADT7:T, and pGBKT7:Lam + pGADT7:T) were co-transfected into competent yeast Y2H Gold. Single colonies were picked and expanded to culture. The resulting culture was then confirmed by PCR. The culture medium containing the bands was spotted on SD / -Trp / -Leu and SD / -Trp / -Leu / -His / -Ade solid media, respectively. The results showed that yeast co-transformed with the pGBKT7:GhGRD1+pGADT7 vectors could grow normally on SD / -Leu / -Trp medium, indicating that the GhGRD1 protein was non-toxic to the yeast strain Y2HGold; at the same time, the yeast strain could not grow on SD / -Trp / -Leu / -His / -Ade medium, indicating that the GhGRD1 protein itself had no self-activation ability ( Figure 8 ).

[0083] To investigate whether GhGRD1 interacts with other proteins involved in drought stress signaling, we used GhGRD1 as bait protein and screened for GhGRD1-interacting proteins using the yeast two-hybrid (Y2H) system. First, the pGBKT7:GhGRD1 vector and the AD library plasmid were co-transformed into competent yeast cells. The yeast culture was then evenly plated onto 50 SD / -Trp / -Leu medium plates to grow single colonies for primary screening. Subsequently, single colonies were picked from the SD / -Trp / -Leu medium and transferred using a sterilized toothpick to SD / -Trp / -Leu / -His / -Ade medium for secondary screening. Positive colonies were then cultured as single colonies. Positive bacterial cultures were then amplified and sequenced using universal primers in the pGADT7 vector. Successful sequences were then BLAST-matched against the cotton database CottonMD (http: / / yanglab.hzau.edu.cn / CottonMD / ) to obtain basic information on potential interacting proteins. Finally, the CDS sequences of the selected genes were cloned into the pGADT7 vector, and each potential interacting protein was verified using a yeast two-hybrid assay. The results showed that yeast co-transformed with pGBKT7+pGADT7:GhGIS (Glabrous inflorescence stems) and pGBKT7+pGADT7:GhMIEL1 (MYB30-interacting E3 ligase 1) vectors grew normally on SD / -Trp / -Leu and SD / -Trp / -Leu / -His / -Ade media, indicating that GhGIS and GhMIEL1 proteins themselves have the ability to activate reporter genes, indicating that the interactions between GhGRD1 and GhGIS and GhMIEL1, respectively, are false positives, and there is no interaction between them ( Figure 9 Yeast co-transformed with pGBKT7:GhGRD1+pGADT7-GhPSBO2 (Photosystem II subunit o-2) and pGBKT7:GhGRD1+pGADT7:GhIBI1 (Impaired in BABA-induced disease immunity 1) vectors were able to grow normally in SD / -Trp / -Leu / -His / -Ade medium, and GhPSBO2 and GhIBI1 proteins did not self-activate, indicating that GhGRD1 interacts with GhPSBO2 and GhIBI1, respectively ( Figure 9 B in ).

[0084] Example 6 Study on the interaction between GhGRD1 and other proteins

[0085] To confirm whether GhGRD1 can interact with GhPSBO2 and GhIBI1 in plants, we GhPSBO2 and GhIBI1 The CDS sequences of the genes were cloned into the 35S::GFP vector, and the corresponding fusion protein expression vectors were successfully constructed. The fusion proteins were expressed in tobacco leaves using Agrobacterium-mediated transient transformation. The transformed tobacco leaves were placed under a Nikon laser confocal microscope for observation, and the subcellular localization of GhPSBO2 and GhIBI1 was analyzed. The experimental results showed that the GFP protein in the empty vector showed normal fluorescence signals in the cell nucleus and cytoplasm. The green fluorescence signals of GhPSBO2:GFP and GhIBI1:GFP overlapped with the red fluorescence signals of H2B:RFP (nuclear localization protein), and green fluorescence signals were also observed in the cytoplasm, indicating that GhPSBO2 and GhIBI1 proteins were simultaneously localized in the cell nucleus and cytoplasm, which is basically consistent with the subcellular localization of GhGRD1 ( Figure 10 This discovery provides important evidence for understanding how GhGRD1 interacts with GhPSBO2 and GhIBI1.

[0086] Since the results of yeast two-hybrid experiments may contain false positives, we chose the bimolecular fluorescence complementation (BiFC) experiment, which is more sensitive and can be performed in plant cells, for additional verification. We constructed the GhGRD1 gene into the pXY104 vector with a cYFP tag and GhPSBO2 and GhIBI1The genes were respectively constructed into the pXY106 vector with nYFP tag. These fusion proteins were expressed in tobacco leaves by Agrobacterium-mediated transformation. In order to ensure the reliability of the experimental results, we selected the interacting protein combination ARP1 (Actin-related protein 1) and ASFT (Aliphatic suberinferuloyl transferase) that the research group has verified as a positive control (Postive). The tobacco leaves were placed under a Nikon laser confocal microscope for observation. The experimental results showed that in the combination of GhGRD1:cYFP and GhPSBO2:nYFP, obvious yellow fluorescence was visible in the nucleus and cytoplasm of tobacco leaves, and there was no fluorescence signal in the control group, indicating that the two proteins interacted in both the nucleus and cytoplasm of plant cells. In the combination of GhGRD1:cYFP and GhIBI1:nYFP, yellow fluorescence was visible in the cytoplasm of tobacco leaves, and there was no fluorescence signal in the control group, indicating that the two proteins interacted in the plant cytoplasm ( Figure 11 ).

[0087] The embodiments described above are only preferred embodiments of the present invention and are only used to explain the present invention, not to limit the scope of implementation of the present invention. For those skilled in the art, it is of course possible to easily make other implementation methods by replacing or changing the technical content disclosed in this specification. Therefore, all changes and improvements made on the principles of the present invention should be included in the scope of the invention of the present invention application.

Claims

1. GhGRD1 Genes and / or GhGRD2 The application of a gene in enhancing drought resistance of a plant is characterized in that: Through overexpression GhGRD1 Genes and / or GhGRD2 Genes to improve drought resistance in plants, the GhGRD1 The CDS sequence of the gene is shown in SEQ ID NO.

1. GhGRD2 The CDS sequence of the gene is shown in SEQ ID NO. 2, and the plant is cotton or Arabidopsis thaliana.

2. Contains GhGRD1 Genes and / or GhGRD2 The application of a recombinant vector of a gene in enhancing drought resistance of cotton or Arabidopsis is characterized in that: By building GhGRD1 Genes and / or GhGRD2 Gene overexpression recombinant vector, to obtain drought-resistant cotton or Arabidopsis plants, the GhGRD1 The CDS sequence of the gene is shown in SEQ ID NO.

1. GhGRD2 The CDS sequence of the gene is shown in SEQ ID NO.

2.

3. A method for enhancing drought resistance of plants, characterized in that: The method is as follows (1) or (2): (1) By increasing the activity of GhGRD1 protein and / or GhGRD2 protein in the target plant, a plant with stronger drought resistance than the target plant is obtained; (2) By promoting the growth of target plants GhGRD1 Genes and / or GhGRD2 Gene expression can produce plants with stronger drought resistance than the target plant; The target plant is cotton or Arabidopsis thaliana, GhGRD1 The CDS sequence of the gene is shown in SEQ ID NO.

1. GhGRD2 The CDS sequence of the gene is shown in SEQ ID NO.2, the amino acid sequence of the GhGRD1 protein is shown in SEQ ID NO.3, and the amino acid sequence of the GhGRD2 protein is shown in SEQ ID NO.

4.

4. The method for enhancing plant drought resistance according to claim 3, wherein Promote the target plant GhGRD1 Genes and / or GhGRD2 The gene is expressed in a manner called overexpression.

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