Application of ghrlp38 protein and its coding gene in regulating drought resistance of cotton

Abstract

This invention belongs to the field of plant molecular biology and molecular breeding technology, specifically relating to the application of a GhRLP38 protein and its encoding gene in regulating drought resistance in cotton. The amino acid sequence of the GhRLP38 protein is shown in SEQ ID No. 2, and the nucleotide sequence of the GhRLP38 protein encoding gene is shown in SEQ ID No. 1. This invention transforms cotton with an overexpression vector of the GhRLP38 protein encoding gene via Agrobacterium-mediated transformation, enhancing the gene's expression level in cotton and thus improving its adaptability to drought and rehydration conditions. Simultaneously, CRISPR / Cas9 gene editing technology is used to obtain… GhRLP38 Gene knockout mutants. Drought stress experiments showed that overexpression... GhRLP38 It can significantly improve the adaptability of cotton to drought and rehydration conditions, while knockout GhRLP38 This reduces the drought resistance of cotton; the results of leaf relative water content measurement showed that the water retention capacity of the overexpressed lines was significantly enhanced. This invention provides a new gene resource for molecular breeding of drought-resistant cotton, which can be used to breed new drought-resistant and stable-yielding cotton varieties.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology and molecular breeding technology, specifically relating to the application of a GhRLP38 protein and its encoding gene in regulating drought resistance in cotton. Background Technology

[0002] Cotton is an important global economic crop, and its growth and development are highly sensitive to water conditions. Drought stress significantly inhibits cotton growth and causes yield loss by affecting physiological processes such as stomatal opening and closing, transpiration, and photosynthesis.

[0003] With climate change leading to more frequent extreme drought events, elucidating the molecular mechanisms of cotton drought resistance, identifying key regulatory genes, and applying them to molecular breeding have become important research directions for cotton genetic improvement.

[0004] Plant receptor-like proteins (RLPs) are a class of important signal sensing and transduction proteins rich in leucine repeat (LRR) domains. They are widely distributed in the cell membrane or membrane-associated regions and are typically involved in the recognition of external stimuli and the transduction of stress signals. Previous studies have shown that various RLPs containing LRR domains play key roles in plant disease resistance, hormone signaling, and responses to abiotic stress.

[0005] However, current research on the function of cotton LRR-RLP genes in synergistic regulation of plant water retention and growth recovery during drought and rehydration stages is still relatively limited, and their application potential in cotton drought-resistant molecular breeding has not been fully explored.

[0006] Therefore, discovering and verifying the drought resistance regulatory function of LRR-RLP genes in cotton is of great significance for molecular breeding of cotton for drought resistance. Summary of the Invention

[0007] In view of the above-mentioned prior art, the purpose of this invention is to provide an application of the GhRLP38 protein and its encoding gene in regulating the drought resistance of cotton.

[0008] To achieve the above objectives, the technical solution adopted by the present invention provides, on the one hand, the application of GhRLP38 protein in regulating the drought resistance of cotton, wherein the amino acid sequence of the GhRLP38 protein is shown in SEQ ID No.2.

[0009] On the other hand, the technical solution of the present invention is to provide GhRLP38 The application of the protein-encoding gene in regulating cotton drought resistance, the nucleotide sequence of the gene encoding the GhRLP38 protein is shown in SEQ ID No. 1.

[0010] Furthermore, the aforementioned GhRLP38The open reading frame (ORF) of the gene is 1458 bp in length, encoding 485 amino acids, with a molecular weight of 53.46 kD; the GhRLP38 protein has an N-terminal signal peptide structure, a transmembrane domain, and multiple leucine repeat sequences.

[0011] Furthermore, the regulation aims to improve the adaptability of cotton under drought and rehydration conditions.

[0012] Furthermore, the improvement is to enhance the expression level of the gene encoding the GhRLP38 protein in cotton.

[0013] Furthermore, the enhanced expression is achieved by transforming cotton with an overexpression vector encoding the GhRLP38 protein using Agrobacterium-mediated transformation.

[0014] On the other hand, the technical solution of the present invention is to provide a method for improving the drought resistance and drought rehydration adaptability of cotton by overexpressing the encoding gene of GhRLP38 protein in cotton; the nucleotide sequence of the encoding gene of GhRLP38 protein is shown in SEQ ID No. 1.

[0015] Furthermore, the overexpression is achieved by transforming cotton with an overexpression vector encoding the GhRLP38 protein using Agrobacterium-mediated transformation.

[0016] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention is the first to identify cotton. GhRLP38 The gene was confirmed through bidirectional genetic experiments involving overexpression and CRISPR / Cas9 gene knockout. GhRLP38 This gene plays a positive regulatory role in the drought and rehydration response of cotton, providing a new genetic resource for molecular breeding of cotton drought resistance.

[0017] 2. This invention enhances... GhRLP38 The expression of the gene in cotton can effectively increase the relative water content of cotton leaves under drought stress, reduce the degree of plant wilting, and show faster growth recovery ability after rehydration, significantly improving the overall adaptability of cotton in the drought-rehydration cycle.

[0018] 3. This invention proposes a cotton molecular breeding strategy that takes into account both drought stress adaptability and growth recovery ability after rehydration, providing important genetic resources and theoretical support for breeding new drought-resistant and stable-yielding cotton varieties, and has good application prospects. Attached Figure Description

[0019] Figure 1 for GhRLP38 Results of drought and ABA-induced responses. A: The effects of drought treatment on transcriptome data. GhRLP38The effect of ABA treatment on expression; B: qPCR detection of the effect of ABA treatment on expression. GhRLP38 Induction of gene expression; analysis of conserved domains of C:GhRLP38. n = 3, **P<0.01.

[0020] Figure 2 Subcellular localization results of GhRLP38 (tobacco cells). bar = 50 μm.

[0021] Figure 3 For silence GhRLP38 This results in reduced drought resistance in cotton. A: Phenotype of positive control plants; B: qPCR detection. GhRLP38 Silent plants and control plants GhRLP38 Gene expression (CLCrVA is the empty vector plasmid control); C: GhRLP38 Phenotypic characteristics of silent plants under drought stress (plants with low expression levels identified were treated with drought for 10 days and photographed). n = 3, **P<0.01.

[0022] Figure 4 For overexpression GhRLP38 Improve cotton drought resistance, knockout GhRLP38 The result is a reduction in the drought resistance of cotton. A: qPCR detection GhRLP38 Overexpression in transgenic cotton leaves GhRLP38 The expression level, n = 3; B: GhRLP38 Edited DNA sequencing results of T1 mutant plants (KO plants carrying) GhRLP38 (homozygous 4 bp deletion mutation); C: GhRLP38 Response of transgenic cotton to PEG6000 stress (wild type (WT) after 24 hours of 15% PEG6000 treatment) GhRLP38 Status of overexpression (OE) and gene editing (KO) plants, Bar = 5 cm); D: Relative water content of WT and transgenic plants after 24 hours of PEG6000 stress, n = 6; E: GhRLP38 Response of transgenic cotton to drought stress (status of WT, OE2 / 3, and KO plants after drought treatment, Bar = 5 cm); F: relative water content of WT and transgenic plants 8 days after drought stress, n = 6; G: cotton leaves were harvested from each group, and leaf weights were recorded at 0, 1, 2, 3, 4, 6, 8, and 10 hours. Leaf water loss rate was calculated based on 0-hour fresh weight, n = 12. **P < 0.01. Detailed Implementation

[0023] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. The following embodiments are for illustrative purposes only and do not constitute a limitation on the scope of the invention.

[0024] Example 1: Screening and Validation of Drought Stress Response Genes in Cotton The test material was upland cotton ( Gossypium hirsutum The L. variety is “Zhongmian Institute 24 (ZM24)”. Cotton seeds were sown in nutrient soil and cultured in an artificial climate chamber under the following conditions: 16 h light / 8 h dark photoperiod, day and night temperatures of 28℃ and 25℃ respectively, and relative humidity of 60%–70%. Seedlings were used for subsequent experiments after reaching the three-leaf stage. High-temperature treatment was applied with day and night temperatures of 38℃ and 32℃ respectively.

[0025] Candidate genes were screened using transcriptome sequencing data from cotton plants subjected to previous drought treatment. RNA sequencing was performed using the Illumina NovaSeq6000 platform, and the genome was aligned to the upland cotton reference genome using HISAT2. Differential expression analysis was performed using DESeq2 software, with |log2FoldChange|≥1 and FDR<0.05 as the screening criteria, resulting in several differentially expressed genes in response to drought. Among them, one gene with unknown function was significantly upregulated under drought treatment conditions. Its nucleotide sequence is shown in SEQ ID No.1, and its amino acid sequence is shown in SEQ ID No.2. This gene was named... GhRLP38 And conduct subsequent analysis of expression patterns and functions.

[0026] GhRLP38 nucleotide sequence: GhRLP38 amino acid sequence: MKKNNHLSPSLPWTTPFIILSILTVHSIKITSAACHVDDGKGLLAFKSGITQDPSGLMLSSWKSGTDCCNWAGINCRENNRVTTISLYGQVDKPDSYLTGTISSSLVKVQNLDGIYFVNLRNI SGPFPDLLFGLPKLLYVYIENSKLSGPLPVNIGKLKQLGALSLEGNRFTGSIPSSISELTQLTELVLGKNEFSGHFPAGIKQIRNLSYLSLEQNKLMGTIPDIFKSFTDLRILRLSHNEFSG KIPESILSLAPKLAYLELGHNRLSGQIPSYLGKFKALDTLDLSWNSFTGVVPKTFSNLTKIFNLDLSHNSLNDPFPQMNVKGIESLDLSYNNFHLKEIPKWVTSSPIIYSLKLAKCGIKMNL DTWKPAETYFYDYIDLSENEITGSPVDLLNRTDDFLVEFKAAGNKLKFDLGKLRIVKTLKELDISRNLVYGKVPAAITGFNKLNVSYNHLCGQLPKNKFPVSSFVGNDCLCGPPLSPCKL (SEQ ID NO:2) To verify GhRLP38 To investigate expression patterns under different adverse conditions, cotton seedlings at the three-leaf-one-heart stage with uniform growth were selected and subjected to different treatments. The normal control group was cultured under normal watering conditions at 28℃ (day) / 25℃ (night); the drought treatment group was treated under 28℃ (day) / 25℃ (night) conditions with watering stopped for 4 days (Dr4) and 8 days (Dr8); the high temperature treatment group was treated under 38℃ (day) / 32℃ (night) conditions with normal watering for 4 days (Ht4); the combined high temperature and drought treatment group was treated under 38℃ (day) / 32℃ (night) conditions with watering stopped for 4 days (HD4); and the recovery treatment group (HDR) was treated under the combined high temperature and drought treatment for 4 days and then recovered to normal watering conditions at 28℃ (day) / 25℃ (night) for 2 days. Leaves were collected immediately after each treatment, flash-frozen in liquid nitrogen, and stored at −80℃ for later use.

[0027] For analysis GhRLP38To assess the response to ABA, cotton seedlings at the three-leaf stage were sprayed with a 100 μmol / L ABA solution, with an equal volume of distilled water serving as a control. Leaf samples were collected at 0 h, 6 h, 12 h, 24 h, 48 h, and 72 h after treatment. Total RNA was extracted using TRIzol reagent, and first-strand cDNA was obtained via reverse transcription. GhUBQ7 As an internal reference gene, real-time quantitative PCR was performed using the SYBR Green fluorescence quantitative PCR kit. Three biological replicates were set up for each treatment, and the relative expression level was calculated using the 2^-ΔΔCt method.

[0028] Using online databases such as SMART, Pfam, and NCBI to analyze GhRLP38 Domain prediction and homology sequence alignment were performed on the encoded protein, and protein structure analysis was conducted on GhRLP38 homologous proteins from Arabidopsis thaliana and cotton.

[0029] Experimental results are as follows Figure 1 As shown. Figure 1 A indicates that, GhRLP38 It can be induced under various abiotic stress conditions. Among them, expression was induced after 4 days of drought treatment (Dr4). GhRLP38 Expression levels only increased slightly, but after 8 days of drought treatment (Dr8), expression levels increased significantly, reaching 24 times that of the normal control group (P < 0.01), indicating that... GhRLP38 It exhibits a strong response to persistent drought stress. During a 4-day (Ht4) high-temperature treatment, GhRLP38 The expression level was slightly increased compared to the control group, while the expression level was higher after 4 days of combined high temperature and drought treatment (HD4). GhRLP38 Expression reached its highest level, 30 times that of the normal control group, and was significantly higher than that of high temperature treatment alone and drought treatment alone (P < 0.01), indicating that the combined stress of high temperature and drought had a significant effect on the expression of high temperature and drought. GhRLP38 It has a synergistic inducing effect. After recovery culture (HDR) GhRLP38 The expression rapidly decreased to near-normal levels, further indicating GhRLP38 It belongs to the typical stress-induced gene expression category.

[0030] Figure 1 B indicates that after treatment with 100 μmol / L ABA, GhRLP38 Expression levels increased rapidly, reaching a peak of 5 times the pre-treatment level at 6 h (P < 0.01), then gradually decreased, recovering to near initial expression levels between 12 and 72 h, indicating that... GhRLP38 It can respond quickly to ABA signals, suggesting that it participates in the ABA-mediated stress signal transduction process.

[0031] Figure 1 C indicates that, GhRLP38The encoded protein is 485 amino acids long, containing an LRRNT domain at the N-terminus and a typical LRR superfamily conserved domain in the middle, belonging to the plant LRR receptor-like protein family. Phylogenetic analysis further showed that GhRLP38 clusters with several LRR receptor-like proteins from Arabidopsis thaliana and cotton in the same branch, exhibiting high sequence homology, indicating that GhRLP38 belongs to the conserved LRR receptor-like protein family.

[0032] Based on the above results, it can be concluded that GhRLP38 It can respond to drought, high temperature, and combined high temperature and drought stress, and is significantly induced by ABA. Its encoded protein has typical LRR receptor protein structural features, suggesting that... GhRLP38 It may participate in the regulation of cotton's response to abiotic stress as a downstream functional factor in the ABA signaling pathway.

[0033] Example 2: Subcellular localization analysis of GhRLP38 Tobacco Benedict ( Nicotiana benthamiana Seeds were sown in nutrient soil and cultured in a greenhouse. Culture conditions were set as follows: a photoperiod of 16 hours light / 8 hours dark, daytime temperature of 25℃, and nighttime temperature of 22℃. When the plants reached the 4-6 true leaf stage (approximately 4 weeks old), leaves with uniform growth and free from pests and diseases were selected for Agrobacterium-mediated transient transformation experiments.

[0034] The full-length coding sequence (CDS) of the GhRLP38 gene was obtained from the cotton genome database. Using cotton cDNA as a template, PCR amplification was performed using 2×Taq Plus Master Mix II (Dye Plus) from Vazyme. GhRLP38 Target gene fragment. The primers used for PCR amplification are as follows: 067- GhRLP38 -SalI-F: AACACGGGGGGACGTCGACATGAAGAAGAACAACCACCTTTCT (SEQ ID NO: 3) 067- GhRLP38 -BamHI-R: GCCCTTGCTCACCATGGATCCCAGTTTACAAGGCGATAATGGAG (SEQ ID NO: 4) The PCR reaction program was as follows: 94℃ pre-denaturation for 10 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 15 s / kb, for a total of 30 cycles; and a final extension at 72℃ for 10 min. After the reaction, the sample was stored at 4℃.

[0035] Using homologous recombination technology, the amplified... GhRLP38 The CDS fragment was ligated into the plant expression vector pWMV067-AADA-EGFP, enabling... GhRLP38 The gene is regulated by the CaMV 35S strong promoter and forms a C-terminal fusion expression structure with the EGFP reporter gene to obtain 35S:: GhRLP38 -EGFP subcellular localization vector. After the recombinant vector was identified and confirmed to be correct by PCR and sequencing, it was transformed into Agrobacterium competent cells. The vector carrying 35S:: GhRLP38 A single colony of Agrobacterium GV3101 expressing the -EGFP vector was inoculated into LB broth containing 50 mg / L kanamycin and 25 mg / L rifampin, and cultured at 28°C with shaking until the bacterial culture reached OD. 600 The value was 0.8. After collecting the bacterial cells, they were resuspended in Agrobacterium infection buffer. The working concentrations of each component in the resuspension were: 10 mmol / L MgCl2, 10 mmol / L MES, and 100 μmol / L acetosyringone. The bacterial concentration was adjusted to OD. 600 The concentration was 0.8, and the bacteria were incubated at room temperature for 2-3 hours to activate their infectivity.

[0036] The bacterial suspension was slowly injected into the mesophyll tissue of *Nicotiana benthamiana* leaves from the underside using a needle-free injector, ensuring thorough saturation. An empty EGFP vector (35S::EGFP) was used as a negative control. Plants infected with *Agrobacterium* were cultured in darkness for 24 hours, followed by 24–36 hours under normal light to allow for full expression of the GhRLP38-EGFP fusion protein before fluorescence observation. EGFP was used as a free green fluorescent protein control, and GhRLP38::EGFP was used as the control. GhRLP38 The expression vector was fused with EGFP. GFP represents the green fluorescence channel, Bright represents the bright-field image, and Merge represents the superposition of the green fluorescence and bright-field image. In the EGFP control group, the fluorescence signal was uniformly distributed throughout the cell, while the fluorescence of the GhRLP38-EGFP fusion protein was mainly distributed in the cell membrane region, indicating that the GhRLP38 protein is localized in the plasma membrane. Figure 2 ).

[0037] Example 3: VIGS Silencing GhRLP38 Reduce cotton drought resistance The upland cotton (Gossypium hirsutum) variety Neolithic K33 was selected as the experimental material for VIGS. Cotton seeds were surface-sterilized and then cultured in a greenhouse. The culture conditions were set as follows: 16 h light / 8 h dark; daytime temperature 28℃, nighttime temperature 22-25℃; relative humidity: 60%-70%. When the cotton seedlings reached the stage where the two cotyledons were fully unfolded but the first true leaf was not yet fully unfolded (approximately 7-10 days), seedlings with consistent growth were selected for the virus-induced gene silencing experiment.

[0038] According to cotton GhRLP38 Gene sequence-specific silencing fragments are designed, and specific regions with low similarity to other homologous genes are selected as silencing target fragments through sequence alignment analysis. GhRLP38 Primers were designed based on nucleotides 120 to 419 of the gene's CDS region to reduce the likelihood of non-specific gene silencing. VIGS- GhRLP38 -F: TGCCTGCAGACTAGTAAAAGGCCTCTTAGCCTTC (SEQ ID NO: 5) VIGS- GhRLP38 -R: ACCTAGGGGCGCGCCTAAACATATAAGAGTTTGGGTAACCC (SEQ ID NO:6) Using cotton cDNA as a template, PCR was used to amplify and obtain GhRLP38 Gene-specific silencing fragments. The obtained... GhRLP38 The silenced fragment is located between 126-425 bp. This was obtained from PCR amplification. GhRLP38 The specific silencing fragment underwent homologous recombination with the pCLCrVA virus-induced gene silencing vector, and the target fragment was inserted into the corresponding position in the pCLCrVA vector (using restriction enzymes SpeI / AscI) to construct... GhRLP38 Gene silencing vector pCLCrVA:: GhRLP38 After the recombinant vector was identified and verified as correct by PCR and sequencing, it was used for subsequent CLCrVA-mediated... GhRLP38 Gene silencing experiments and verification of drought resistance function.

[0039] The correct recombinant plasmid (pCLCrVA::) will be verified. GhRLP38The empty vector control (pCLCrVA::00), positive control (pCLCrVA::Albino), and viral genome plasmid (pCLCrVB) were introduced into competent cells of *Agrobacterium tumefaciens* strain GV3101 via heat shock. Verified single clones were inoculated into LB broth containing 50 mg / L kanamycin and 25 mg / L rifampin and cultured with shaking at 28°C. The bacterial culture was cultured to OD... 600 The bacterial cells were collected by centrifugation at a pH of 1.5 and resuspended in infection buffer. The infection buffer formulation was: 10 mmol / L MgCl2, 10 mmol / L MES, 100 μmol / L acetosyringone, pH adjusted to 5.6. The OD of the Agrobacterium suspension was adjusted. 600 Adjust the pH to 1.5 and allow it to incubate at room temperature in the dark for 2-3 hours to induce growth. Mix the appropriate bacterial suspension (pCLCrVA::) at a 1:1 volume ratio according to experimental needs. GhRLP38 Mix pCLCrVA::00 and pCLCrVA::Albino with equal volumes of pCLCrVB, and let stand at room temperature for 3 hours before injection. Gently prick the underside of the leaf with the tip of a 1 mL syringe needle and inject through the small opening (Note: The index finger of the hand holding the leaf should lightly block the needle opening on the leaf surface to prevent injection outside the leaf; excessive force will damage the leaf). Place the grafted plants in an insect-proof greenhouse and culture in darkness at 22-25℃ for 24 hours, then transfer to light conditions for continued cultivation (16 hours light / 8 hours darkness; daytime 28℃, nighttime 22-25℃; relative humidity: 60%-70%). When the plants reach one month of age, the albino phenotype begins to appear. At this time, pCLCrVA::00 is then injected. GhRLP38 Tender leaf tissues were taken from the plant and qPCR silencing efficiency was detected.

[0040] Figure 3 B indicates that the silencing efficiency of some plants reached over 70% (CLCrVA:: GhRLP38 Plants 1, 5, and 7 can be used for subsequent verification of drought resistance. Figure 3 A indicates that the CLCrVA system can effectively induce gene silencing in cotton, proving the reliability of the VIGS system. The control plants and those with low expression levels were compared. GhRLP38 Silent plants were treated with drought for 10 days, and the results were as follows: Figure 3 As shown in C: Under normal conditions GhRLP38 Silent plants showed no obvious growth defects; however, under drought stress conditions... GhRLP38 Silent plants exhibited a more severe wilting phenotype. Therefore, it can be concluded that... GhRLP38 Gene silencing reduces cotton's tolerance to drought stress, indicating that... GhRLP38 It plays a positive regulatory role in the drought resistance response of cotton.

[0041] Example 4: Overexpression and Knockout GhRLP38 Verify its drought resistance function 1. Creation of transgenic materials: The upland cotton (Gossypium hirsutum) variety Neolithic K33 was selected as the genetic transformation recipient material. The transgenic material was obtained using an Agrobacterium-mediated genetic transformation system. GhRLP38 Overexpression plants ( GhRLP38 -OE) and GhRLP38 Gene knockout plants ( GhRLP38 -KO), with unconverted wild-type plants (WT) as a control. All materials were cultured under greenhouse conditions: 16 h light / 8 h dark; daytime 28°C, nighttime 22-25°C; relative humidity: 60%-70%. When the plants reached the three- to four-leaf stage, they were used for subsequent transgenic material creation.

[0042] According to cotton GhRLP38 Gene CDS sequence-specific primers were designed (primers are the same as in Example 2), and cotton cDNA was used as a template for amplification. GhRLP38 Complete encoded sequence. Using homologous recombination, ... GhRLP38 The gene CDS fragment was ligated into the plant expression vector pWMV067-AADA-EGFP to construct a CaMV 35S promoter-driven expression vector. GhRLP38 Overexpression vector (construction method and amplification conditions are the same as in Example 2): 35S:: GhRLP38 After the recombinant plasmid was identified by PCR and verified by sequencing, it was transformed into the Agrobacterium EHA105 recipient strain for cotton genetic transformation.

[0043] according to GhRLP38 Gene sequence-specific sgRNA target sites are designed. Sites located in... GhRLP38 Off-target analysis of the specific target sequences at the front end of the coding region was performed using an online tool (http: / / crispr.hzau.edu.cn / CRISPR2 / ) to screen for highly specific sgRNA sequences: sgRNA1: 5'-TAAAAGTCCAAAATCTAGA-3' (SEQ ID NO: 7) sgRNA2: 5'-TTACCTGTGAATATTGGTA-3' (SEQ ID NO:8) The designed sgRNA sequence was ligated into the CRISPR / Cas9 gene editing vector WMC016 to construct... GhRLP38 Gene knockout vector: CRISPR / Cas9- GhRLP38After confirmation by sequencing, the recombinant vector was transformed into Agrobacterium strains for cotton genetic transformation. Agrobacterium-mediated transformation of cotton was performed. The vector carrying pWMV067- GhRLP38 Overexpression vector or WMC016- GhRLP38 Agrobacterium strains with the vector knocked out were cultured on LB solid medium containing 50 mg / L kanamycin and 25 mg / L rifampin, and single colonies were picked for expansion culture. The bacterial cells were collected and resuspended to an appropriate concentration (OD600 of 0.6–0.8) in resuspension solution (10 mmol / L MES, 10 mmol / L MgCl2, 200 μmol / L AS, pH 5.7).

[0044] Using the shoot tip meristem of sterile upland cotton 'Xinshi K33' seedlings as the transformation recipient material, resistant regenerated plants were obtained through Agrobacterium infection, co-culture, sterilization screening, and regeneration culture. After infection with Agrobacterium suspension containing 200 μmol / L acetylsyringone (AS) for 15–20 min, the shoot tips were inoculated onto MS solid co-culture medium containing 200 μmol / L AS and cultured in the dark at 25°C for 2–3 days. Subsequently, they were transferred to MS medium containing 300–500 mg / L cephalosporin (Cef) for sterilization culture for 3–5 days, and then transferred to MS selection medium containing 200 mg / L spectinomycin and 200–300 mg / L Cef, and cultured at 28°C under 16 h light / 8 h dark conditions for 2–3 weeks, with subculture every 7–10 days. After obtaining resistant shoots, they were transferred to MS rooting medium containing 20 mg / L spectinomycin, 100 mg / L Cef, and 0.1–0.2 mg / L NAA and cultured for 1–2 weeks. Once a complete root system had formed, the seedlings were cultured for 3–5 days to obtain resistant regenerated plants. These were then further propagated to obtain stable genetic material: overexpression lines (OE2, OE3) and homozygous knockout mutants (KO). Figure 4 A, 4B).

[0045] 2. Identification of drought resistance phenotypes: Selecting WT, GhRLP38 -OE and GhRLP38 -KO cotton seeds were subjected to PEG6000-simulated drought treatment. Seeds were sown in boxes containing only vermiculite and cultured under a film until the seed coat completely detached. They were then carefully transferred to a Hoagland nutrient solution culture system for acclimatization. Subsequently, PEG6000 was added to simulate water stress. Treatment conditions: Control group: normal nutrient solution culture; drought treatment group: 15% PEG6000 solution added to simulate drought stress. Treatment time was set as: 0 h; 12 h; 24 h. During treatment, the following were observed and recorded: leaf wilting degree, root growth status, and plant growth changes. It was observed that the OE line showed the least wilting, followed by the wild type, and the KO line showed the most severe wilting. Figure 4C). Simultaneously, leaf samples were collected for relative water content determination: under normal culture conditions. GhRLP38 Changes in expression do not affect the moisture state of cotton; under drought conditions, GhRLP38 -OE cotton has enhanced water retention capacity. GhRLP38 -KO cotton's water retention capacity decreases ( Figure 4 D).

[0046] Select seedling stage WT, GhRLP38 -OE and GhRLP38 -KO cotton plants with uniform growth were cultured in a greenhouse under the following conditions: 16 h light / 8 h darkness; daytime temperature 28℃, nighttime temperature 22~25℃; relative humidity 60%~70%. Natural water loss was observed, and the results were consistent with those of hydroponics: the OE strain showed the least wilting, followed by the WT strain, and the KO strain showed the most severe wilting. Figure 4 E). The relative water content of leaves after natural water loss was measured, and the results showed that the OE line was significantly higher than the WT line, while the KO line was significantly lower than the WT line. Figure 4 F). Leaf water loss rate was measured continuously for 10 hours: with the increase of time, the water loss rate of OE, WT and KO lines gradually increased, but the water retention capacity of OE line was better than that of WT line, and the water retention capacity of KO line was weaker than that of WT line. Figure 4 G).

[0047] The above results indicate that GhRLP38 It is a positive regulatory gene for drought resistance in cotton. Overexpression of this gene can effectively improve the drought resistance of cotton and has important breeding application value.

[0048] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of GhRLP38 protein in regulating drought resistance in cotton, characterized by, The amino acid sequence of the GhRLP38 protein is shown in SEQ ID No.

2.

2. The application of the gene encoding the GhRLP38 protein in regulating drought resistance in cotton, characterized by: The nucleotide sequence of the gene encoding the GhRLP38 protein is shown in SEQ ID No.

1.

3. The application according to claim 2, characterized in that, The aforementioned regulation aims to improve the adaptability of cotton under drought and rehydration conditions.

4. The application according to claim 3, characterized in that, The improvement refers to enhancing the expression level of the gene encoding the GhRLP38 protein in cotton.

5. The application according to claim 4, characterized in that, The enhanced expression was achieved by transforming cotton with an overexpression vector encoding the GhRLP38 protein using Agrobacterium-mediated transformation.

6. A method for improving the drought resistance and adaptability to drought and rehydration in cotton, characterized in that, The gene encoding the GhRLP38 protein was overexpressed in cotton; the nucleotide sequence of the gene encoding the GhRLP38 protein is shown in SEQ ID No.

1.

7. The method according to claim 6, characterized in that, The overexpression was achieved by transforming cotton with an overexpression vector encoding the GhRLP38 protein using Agrobacterium-mediated transformation.

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