Application of GhMATE27 in regulating upland cotton fiber quality and drought resistance of cotton plants

Abstract

This invention belongs to the field of biotechnology, specifically relating to the application of GhMATE27 in regulating upland cotton fiber quality and drought resistance in cotton plants. This invention proposes the role of GhMATE27 in regulating upland cotton fiber quality and drought resistance in cotton plants, improving drought resistance in cotton plants by silencing GhMATE27 and improving upland cotton fiber quality by knocking out GhMATE27.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of GhMATE27 in regulating the fiber quality of upland cotton and the drought resistance of cotton plants. Background Technology

[0002] Cotton is one of the world's most important economic crops and the largest source of natural fiber. As a major economic crop and raw material for my country's textile industry, cotton has formed a vast industrial chain. Statistics show that my country's average annual cotton production and consumption account for approximately 21% and 26% of the global total, respectively. However, cotton production capacity is severely insufficient, resulting in an annual cotton consumption gap of approximately 2 million tons. Simultaneously, the quality of cotton fiber produced in my country is relatively low, with a severe shortage of high-quality cotton fiber and a significant surplus of low-quality cotton fiber. The high-end raw cotton market largely relies on imports. This severe lack of mid-to-high-end raw cotton poses a significant challenge to the cotton industry. Furthermore, my country's main cotton-growing areas are the Yangtze River region, the Yellow River region, and the Xinjiang region, with Xinjiang being the primary cotton-producing area. In 2023, Xinjiang's output accounted for approximately 91% of the national total. The Xinjiang region is chronically arid with little rainfall, and water scarcity and drought caused by high temperatures have become significant factors limiting local cotton production. Therefore, improving cotton quality and drought resistance has become a key focus of cotton breeding in my country to better meet production and living needs.

[0003] Flavonoids generally refer to a series of compounds consisting of two benzene rings with phenolic hydroxyl groups linked by three central carbon atoms. Chemically, flavonoids have a three-ring structure with a C6-C3-C6 framework. Based on the oxidation degree of the central heterocycle, flavonoids are generally classified into seven subclasses, including flavones, isoflavones, flavonols, flavanones, flavanols, chalcones, and anthocyanins. In plants, flavonoids are typically found in organs such as flowers, leaves, and seeds. They usually accumulate in plant cell vacuoles in the form of glycosides. Flavonoids play a crucial role in many life activities of plants, including growth, development, and maturation. They not only participate in pigment synthesis to regulate the color of plant organs but also regulate root growth, signal transduction, auxin transport, and participate in allelopathic effects. Furthermore, flavonoids help plants resist various biotic and abiotic stresses.

[0004] Cotton fiber, a crucial product in cotton production, directly determines the output value and profitability of cotton production through its yield and quality. Therefore, improving cotton fiber quality has always been a primary goal of cotton breeding. While there is considerable research on cotton fiber quality, studies linking flavonoids to it are limited. Some studies have found that exogenous application of high concentrations of naringenin and dihydrokaempferol significantly inhibits cotton fiber growth and development, particularly elongation, leading to reduced fiber quality. Similarly, introducing the F3H-RNAi fragment into brown cotton T586 resulted in naringenin accumulation inhibiting fiber elongation. Another study found that the flavonoid rutin significantly promotes cotton fiber initiation, while naringenin inhibits fiber elongation in the later stages of fiber development. Therefore, the effects of different types of flavonoids on cotton fiber quality at different stages of fiber development may be contradictory, requiring more systematic and comprehensive research. Currently, research reports on the effects of flavonoids on cotton drought resistance are limited, necessitating further in-depth investigation. Therefore, improving cotton fiber quality and drought resistance by altering flavonoid content is a highly promising approach. However, the molecular mechanisms regulating fiber quality and drought resistance in upland cotton remain unresolved. In breeding practice, available gene resources regulating upland cotton fiber quality and drought resistance are extremely scarce, making the role of these gene resources unclear. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides the application of GhMATE27 in regulating the fiber quality of upland cotton and the drought resistance of cotton plants.

[0006] The application of GhMATE27 in regulating upland cotton fiber quality and cotton plant drought resistance, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] Preferably, the expression of GhMATE27 is suppressed to improve the quality of the upland cotton fiber.

[0008] Preferably, the expression of GhMATE27 is inhibited to improve the drought resistance of the cotton plant.

[0009] Preferably, the expression of GhMATE27 is suppressed by either virus-induced gene silencing or CRISPR-Cas9.

[0010] Preferably, the method for virus-induced gene silencing involves amplifying the GhMATE27 fragment using the primers shown in SEQ ID NO.2-3, inserting the GhMATE27 fragment into the pTRV2 vector to obtain the VIGS vector, transforming the VIGS vector with Agrobacterium tumefaciens, and then infecting it with the virus to silence GhMATE27.

[0011] Preferably, the CRISPR-Cas9 method involves assembling the sgRNA sequence shown in SEQ ID NO.15-16 into the vector pYLCRISPR / Cas9P35S-N to obtain the CRISPR-Cas9 vector, and then transforming the CRISPR-Cas9 vector with Agrobacterium to knock out GhMATE27.

[0012] Preferably, the upland cotton is upland cotton TM-1.

[0013] A method for improving the quality of upland cotton fibers involves assembling the sgRNA sequence shown in SEQ ID NO.15-16 into the vector pYLCRISPR / Cas9P35S-N to obtain a CRISPR-Cas9 vector, and transforming the CRISPR-Cas9 vector with Agrobacterium to knock out GhMATE27, thereby improving the quality of upland cotton fibers.

[0014] Preferably, the upland cotton is upland cotton TM-1.

[0015] A method for improving the drought resistance of cotton plants involves amplifying the GhMATE27 fragment using primers shown in SEQ ID NO.2-3, inserting the GhMATE27 fragment into a pTRV2 vector to obtain a VIGS vector, and then transforming the VIGS vector with Agrobacterium and infecting it with a virus to silence the GhMATE27 fragment, thereby improving the drought resistance of the cotton plants.

[0016] Compared with the prior art, the advantages of the present invention are:

[0017] This invention proposes the role of GhMATE27 in regulating the fiber quality and drought resistance of upland cotton plants. It improves the drought resistance of cotton plants by silencing GhMATE27 and improves the fiber quality of upland cotton by knocking out GhMATE27. Attached Figure Description

[0018] Figure 1 Accumulation profiles of flavonoids in different tissues of 383 upland cotton materials.

[0019] Figure 2 This is a genome-wide association study based on metabolites.

[0020] Figure 3 For protoplast subcellular localization experiments.

[0021] Figure 4 Drought tolerance experiment under VIGS treatment.

[0022] Figure 5Phenotypic verification experiments for transgenic knockout materials. Detailed Implementation

[0023] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods.

[0024] Example 1

[0025] Analysis of the accumulation pattern of flavonoids in upland cotton petals

[0026] To analyze the effects of upland cotton petal flavonoids on cotton fiber quality and drought resistance, we used 383 upland cotton varieties as materials and planted them in Anyang City, Henan Province. All materials were planted in the experimental field in sequence, with two replicates. Petal samples were taken on the day of flowering, and the day of flowering was recorded as 0DPA. The samples were then quick-frozen with liquid nitrogen and stored in a -80℃ freezer.

[0027] Targeted metabolomics assay

[0028] Cotton petals from flowers at 0DPA (0% fresh weight) were used as samples, with each sample weighing 0.3-0.5 grams fresh, and two biological replicates were set up. Then, using targeted metabolomics, the content of 16 flavonoid compounds in cotton petals was detected by liquid chromatography-mass spectrometry.

[0029] (1) Sample preparation: Fresh material was placed in 2 mL EP tubes and immediately flash-frozen in liquid nitrogen and stored in an ultra-low temperature freezer at -80℃. The day before sample preparation, the corresponding tissue material was freeze-dried for 16 h, and 0.100 g was accurately weighed using a balance. The powder was ground into powder using a ball mill and added to the extraction solution at a ratio of 0.1 g / mL. The mixture was shaken on a vortex mixer for 15 min, centrifuged at 15000 rpm for 30 min at 4℃, filtered through a 0.22 μm filter membrane, and 150 μL to 200 μL was placed into vials for analysis using a QTrap4000 liquid chromatography-mass spectrometry (LC-MS) system. The extraction solution was 80% methanol-water (v / v) containing salicylic acid as an internal standard.

[0030] (2) Instrumental Detection: This experiment uses an ESI ion source. The retention times (RT / s) of the detected Q1 precursor ion and Q3 fragment ion are compared with the retention times of the standard to perform qualitative analysis of the detected ions. If the retention times are the same, they can be identified as the same compound. The peak areas are integrated, and the concentration of the detected ions is absolutely quantified based on the standard curve. The mobile phases used in the experiment are: Phase A is a 0.05% (v / v) formic acid aqueous solution, and Phase B is a 0.05% (v / v) formic acid acetonitrile. Flavonoid standards are used as external standards, and a concentration gradient is set to create a standard curve. Simultaneously, eucalyptol in the extract is used as an internal standard to eliminate machine noise and reduce instrument error.

[0031] Results: A total of 16 types of flavonoids belonging to 7 classes were detected in the petals. Flavonols were found to be present in higher concentrations, while other flavonoids were present in lower concentrations, indicating that flavonol metabolism is more active in the petals. Furthermore, a clear population-specific pattern was observed in the accumulation of flavonols. Figure 1 As shown.

[0032] Example 2

[0033] Discovering key genes regulating flavonoid content in cotton petals

[0034] (1) Descriptive statistical analysis: By sorting out the data distribution characteristics of all detected flavonoids in the population and performing logarithmic transformation on the absolute content data of each flavonoid, it was found that the flavonoid content in the petals conforms to a normal or approximately normal distribution, which is suitable for subsequent GWAS analysis.

[0035] (2) Genome-wide association analysis based on metabolites: Targeted metabolomics analysis of flavonoid metabolites in petals was performed on 383 natural populations of upland cotton, yielding a large amount of data, which was then subjected to mGWAS with resequencing genomic data. We used 1,076,652 high-quality SNPs, ensuring minor allele frequencies >0.05 and deletion rates <0.2. Simultaneously, a mixed linear model was used for mGWAS, identifying 3778 SNPs highly associated with flavonoids above the intermediate significance threshold. We also plotted a Manhattan plot using petal catechin content as a target trait, and performed LD block analysis and haplotype analysis, such as... Figure 2 As shown.

[0036] (3) Candidate Gene Identification: We identified a total of 50 quantitative trait loci (QTLs), containing 235 candidate genes associated with flavonoid content in specific tissues. Among these was a gene involved in the extrusion transport of multidrugs and toxic compounds, designated GhMATE27. Further investigation into the impact of significant SNPs on the function of each gene revealed that a single SNP on GhMATE27 caused premature translation termination, significantly affecting protein structure and function. Therefore, GhMATE27 was selected as a candidate gene for subsequent functional validation.

[0037] Example 3

[0038] Functional verification of GhMATE27

[0039] The gene number of GhMATE27 is Gh A06G036100, and its nucleotide sequence is: ATGGATGATA.

[0040] CCCATAGGAACAGTGACGAATACCATCAACCTTTACTAACAGAACCAAGT

[0041] TCAAATGATGAAGCAACTATATTGAGAGATGAAGATGGTGAAGAAGGAAA

[0042] GGACTTAAGTACAAGGCTATGGATCGAAACAAAGAAGCTATGGGTCATCG

[0043] TAGGACCTTCAATCATCAGTCGTGTTGCTGGTTACTCCATGAACATTATAA

[0044] CCCAAGCTTTTGCTGGTCACCTTGGGTGATGTTGAACTTGCTGCTATTTCAA

[0045] TAGCCAATACGGTCATCGTTGGCTTCAACTTTGGCTTCCTACTAGGGATGG

[0046] CGAGTGCTTTAGAAACCCTTTGTGGACAAGCTTTTGGTGCTAAACAATAC

[0047] CACATGTTAGGCATTTACATGCAAAGATCATGGATCGTTTTACTCCTTTGTT

[0048] GTTTCTTGTTGTTACCCTTTTACGTATTTGCTACCCCCGTTTTAAAGCTATTA

[0049] GGACAGCCGGACGATGTGGCGGAGATGTCGGGGGTGGCGGCGATTTGGA

[0050] TGATACCTTTGCACTTTAACTTTGCGTTTCAGTTCCCATTACAAAGGTTCTT

[0051] GCAAAGCCAGTCGAAGACTCTGGTGTTAGCCTATGCCTCGTTTGGTGCTTT

[0052] AGGGGTGAATGTGTTGACGAGTTGGGTGTTCGTAAACGTGTTGGATTGGG

[0053] GTGTGATTGGTGCTTCGTTGGCGTTGGATATTTCGTGGTGGGTTGGGTCTT

[0054] TGGGGCTTTATAGCTACACTGTCCTCGGTGGCTGTCCTTTGTCATGGACCG

[0055] GGTACTCGATGGAAGCATTTAATGGCTTATGGGAGTTTCTTAAACTCTCTG

[0056] CTTCCTCTGGAGTCATGCTATGGTACATCAAAACAACATACATATATAAATA

[0057] TCTTGGAACTTGGTACTATCGGATTCTGATATTGATGACTGGATACTTGTCA

[0058] AATGCAACTTTGGCTGTGGATGCCTTATCCGTTTGTATGAGTATCAATGGTT

[0059] GGGAGCTTATGATTCCTCTGGCTTTCCTGGCTGCTACTGGAGTAAGGGTGG

[0060] CAAATGAGCTAGGAGCAGGCAATTGGAAAGGGGCGAAATTTGCTACAAA

[0061] GGTGTCAATCGTGCAATCGACAATCGTTGGAGTATTCTTCTGTATAATAGT

[0062] GTTGGTACTTCGTGATAAAGTGGCATTGATATTCACATCCAGCAATGATGT

[0063] CCTTGAAGAAGTTGATAAGCTGTCTTACCTGTTAGGCGTCACCATTCTACT

[0064] CAACAGTGTTCAGCCAGTCTTTATCAGGAGTGGCAATTGGATCAGGGTGGC

[0065] AAGCAACGGTGGCTTACATAAACTTGGGTTGCTATTATATTATTGGACTCCC

[0066] TCTTGGGATTTTGATGGGATGGGTTTTCAAGTTGGGTGTCTCGGGAATTTG

[0067] GGGTGGGATGATCTTTGGAGGAACAGCTATTCAGACAGTGATTTTGGCTAT

[0068] ATTCACAATTCGAAGAGATTGGAAAAAGGAGGCTGAAAAGGCTAAGCAG

[0069] AGAGTTGGAAGGTGGGCGAAGTGA, denoted as SEQ ID NO.1.

[0070] I. Subcellular localization of protoplasts

[0071] By binding the GFP tag to the GhMATE27 protein and then to the localization of the γTIP-RFP tag, it was found that this protein is localized on the vacuolar membrane, such as... Figure 3 As shown.

[0072] II. VIGS Experiment

[0073] 1. Construction of VIGS carrier

[0074] (1) Cloning the target gene fragment: The target gene fragment was amplified using PCR technology. The target gene was GhMATE27, and the coding region was selected to be approximately 300-500 bp in length.

[0075] The amplification primers are as follows:

[0076] F: CGGGAATTCAGTGTTGGTACTTCGTGATAAAGT, recorded as SEQ ID NO.2;

[0077] R: AAAGGTACCATCACTGTCTGAATAGCTGTTCC, denoted as SEQ ID NO.3;

[0078] Target gene fragment: AGTGTTGGTACTTCGTGATAAAGTGGCATTGATATTCA CATCCAGCAATGATGTCCTTGAAGAAGTTGATAAGCTGTCTTACCTGTTAGGCGTCACCATTCTACTCAACAGTGTTCAGCCAGTCTTTATCAGGAGTGGCAATTGGATCAGGGTGGCAAGCAACGGTGGCTTA CATAAACTTGGGTTGCTATTATATTATTGGACTCCCTCTTGGGATTTTGATGGGATGGGTTTTCAAGTTGGGTTCTCGGGAATTTGGGGTGGGATGATCTTTGGAGGAACAGCTATTCAGACAGTGAT, recorded as SEQ ID NO.4.

[0079] (2) Construction of VIGS vector: The cloned target gene fragment was inserted into the pTRV2 vector using DNA recombination technology to obtain the VIGS vector, and sequencing was performed to verify that the vector was constructed correctly.

[0080] (3) Transformation of host cells: The universal vector pTRV1 and the constructed VIGS vector were transformed into Escherichia coli and identified.

[0081] 2. Viral Reproduction and Infection

[0082] (1) Transformation of Agrobacterium: pTRV1 vector and VIGS vector were extracted from Escherichia coli and transformed into Agrobacterium GV3101.

[0083] (2) Virus propagation: The Agrobacterium solution containing pTRV1 was resuspended in VIGS buffer to obtain mixture one, and the Agrobacterium solution containing VIGS vector was resuspended in VIGS buffer to obtain mixture two. Mixture one and mixture two in equal proportion and incubate at 28℃ for 3h to obtain virus suspension. The VIGS buffer contains 10 mmol / L MgCl2, 10 mmol / L MES and 200 μmol / L AS, and the pH is 5.6.

[0084] (3) Viral infection: The virus suspension obtained from propagation was injected into the cotyledons of 1-week-old upland cotton TM-1 seedlings. The virus entered the plant cells and infected them, thereby silencing the target gene.

[0085] Plants transformed with TRV:CLA1 (GhCLA1) served as a positive control in this experiment. TRV:00 (empty vector) plants served as a negative control. Three parallel experiments were set up for each experimental group.

[0086] Virus-induced gene silencing (VIGS) reduced GhMATE27 expression, and the gene-silenced material showed severe wilting under drought conditions with significantly increased H2O2 and malondialdehyde (MDA) levels compared to the control group. This indicates that GhMATE27 expression can indeed improve the drought resistance of cotton plants. Figure 4 As shown.

[0087] III. Transgenic Experiments

[0088] 1. Construct the CRISPR-Cas9 vector, following the method described in Ma et al., 2015, Molecular Plant. The specific method is as follows:

[0089] (1) Synthesis of sgRNA

[0090] Using pYLgRNA-AtU3b as a template, PCR was performed using primers UF and sgRNA-xR. The resulting product was denoted as product A from the first round of PCR. In sgRNA-xR, x represents 1 or 2.

[0091] Using pYLgRNA-AtU6-29 as a template, PCR was performed using primers sgRNA-xF and gR-R. The resulting product was designated as product B from the first round of PCR. In sgRNA-xF, x represents either 1 or 2.

[0092] Using first-round PCR product A and first-round PCR product B as templates, complete sgRNAs were cloned using primer pairs Pps-GGL and Pgs-GG2R, and primer pairs Pps-GG2F and Pgs-GGR.

[0093] The nucleotide sequence of the template pYLgRNA-AtU3b is: GTTTTAGAGCTAGAAATAGCA AGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGAATTTTCCTCCGTTTTACCTGTGGAATCGGCAGCAAAGGATTTACTTTAAATTTTTTCTTATGCAGCCTGTGATGGATAACTGAATCAAACAAATGGCGTCTGGGTTTAAGAAGATCTGTTTTGGCTATGTTGGACGAAACAAGTGAACTTTTAGGATCAACTTCAGTTTATATATGGAGCTTATATCGAGCAATAAGATAAGTGGGCTTTTTATGTAATTTAATGGGCTATCGTCCATAGATTCACTAATACCCATGCCCAGTACCCATGTATGCGTTTCATATAAGCTCCTAATTTCTCCCACATCGCTCAAATCTAAACAAATCTTGTTGTATATATAACACTGAGGGAGCAACATTGGTCA, denoted as SEQ ID NO.17.

[0094] The nucleotide sequence of the template pYLgRNA-AtU6-29 is: GTTTTAGAGCTAGAAATA GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGAATTTTCCTCCGTTTTACCTGTGGAATCGGCAGCAAAGGAAAATATCAGAGATCTCTTACAGTTAGTTTCGTTCTTAATCCAAACTACTGCAGCCTGACAGACAAATGAGGATGCAAACAATTTTAAAGTTTATCTAACGCTAGCTGTTTTGTTTCTTCTCTCTGGTGCACCAACGACGGCGTTTTCTCAATCATAAAGAGGCTTGTTTTACTTAAGGCCAATAATGTTGATGGATCGAAAGAAGAGGGCTTTTAATAAACGAGCCCGTTTAAGCTGTAAACGATGTCAAAAACATCCCACATCGTTCAGTTGAAAATAGTAGCTCTGTTTATATATTGGTAGAGTCGACTAAGAGATTG, denoted as SEQ ID NO.18.

[0095] U-F: CTCCGTTTTACCTGTGGAATCG, denoted as SEQ ID NO.5;

[0096] sgRNA-1R: TAGGAACAGTGACGAATACCAATCTCTTAGTCGACT, denoted as SEQ ID NO.6;

[0097] sgRNA-1F: GTATTCGTCACTGTTCCTAGTTTTAGAGCTAGAAAT, denoted as SEQ ID NO.7;

[0098] sgRNA-2R: TCTTCACCATCTTCATCTCTGACCAATGTTGCTCC, denoted as SEQ ID NO.8;

[0099] sgRNA-2F: GAGATGAAGATGGTGAAGAGTTTTAGAGCTAGAAAT, denoted as SEQ ID NO.9;

[0100] gR-R: CGGAGGAAAATTCCATCCAC, denoted as SEQ ID NO.10;

[0101] Pps-GGL: TTCAGAGGTCTCTCTCGACTAGTATGGAATCGGCAGCAAAG G, denoted as SEQ ID NO.11;

[0102] Pgs-GG2R: AGCGTGGGTCTCGTCAGGGTCCATCCACTCCAAGCTC, denoted as SEQ ID NO.12;

[0103] Pps-GG2F: TTCAGAGGTCTCTCTGACACTGGAATCGGCAGCAAAGG, denoted as SEQ ID NO.13;

[0104] Pgs-GGR: AGCGTGGGTCTCGACCGACGCGTATCCATCCACTCCAAGC TC, denoted as SEQ ID NO.14.

[0105] The cloned sgRNAs include sgRNA-1 and sgRNA-2.

[0106] The nucleotide sequence of sgRNA-1 is: TTCAGAggtctcTctcgACTAGTATGGAATCGGC AGCAAAGGAAAATATCAGAGATCTCTTACAGTTAGTTTCGTTCTTAATCCAAACTACTGCAGCCTGACAGACAAATGAGGATGCAAACAATTTTAAAGTTTATCTAACGCTAGCTGTTTTGTTTCTTCTCTCTGGTGCACCAACGACGGCGTTTTCTCAATCATAAAGAGGCTTGTTTTACTTAAGGCCAATAATGTTGATGGATCGAAAGAAGAGGGCTTTTAATAAACGAGCCCGTTTAAGCTGTAAACGATGTCAAAAACATCCCACATCGTTCAGTTGAAAATAGTAGCTCTGTTTATATATTGGTAGAGTCGACTAAGAGATTGGTATTCGTCACTGTTCCTAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGACCCTGACGAGACCCACGCT, denoted as SEQ ID NO.15.

[0107] The nucleotide sequence of sgRNA-2 is: TTCAGAGGTCTCTCTGACACTGGAATCGGC AGCAAAGGATTTACTTTAAATTTTTTCTTATGCAGCCTGTGATGGATAACTGAATCAAACAAATGGCGTCTGGGTTTAAGAAGATCTGTTTTGGCTATGTTGGACGAAACAAGTGAACTTTTAGGATCAACTTCAGTTTTATATATGGAGCTTATATCGAGCAATAAGATAAGTGGGCTTTTTATGTAATTTAATGGGCTATCGTCCATAGATTCACTAATACCCATGCCCAGTACCCATGTAT GCGTTTCATATAAGCTCCTAATTTCTCCCACATCGCTCAAATCTAAACAAATCTTGTTGTATATATAACACTGAGGGAGCAACATTGGTCAGAGATGAAGATGGTGAAGAGTTTTAGAGCTA GAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGATACGCGTCGGTCGAGACCCACGCT, denoted as SEQ ID NO.16.

[0108] (2) Connecting sgRNA to vector

[0109] The above sgRNA sequence was assembled into the vector pYLCRISPR / Cas9P35S-N using the GoldenGate method.

[0110] (3) Transformation and Screening

[0111] The ligated vector was transformed into competent E. coli, and positive clones were selected by resistance screening. Plasmids were extracted and sequenced for verification. If the sequencing was correct, the recombinant vector, namely the CRISPR-Cas9 vector, was obtained.

[0112] 2. Screening and identification of transgenic cotton plants

[0113] (1) Creation of transgenic materials

[0114] The recombinant vector was transformed into Agrobacterium strain LBA4404, and then the CRISPR-Cas9 vector was transformed into recipient cotton using the Agrobacterium transformation method.

[0115] (2) Gene editing identification

[0116] The target site was amplified by PCR and identified by sequencing. Mutations in the target sequence were identified as mutant materials, and mutant materials Ghmate27-L02 and Ghmate27-L16 were obtained.

[0117] (3) Phenotypic measurement of mutants

[0118] The creation of transgenic knockout materials was successfully completed using the CRISPR-Cas9 system. Firstly, the growth phenotype of the transgenic materials showed no significant difference compared to wild-type (WT) materials. Secondly, in three parallel experiments comparing the fiber length of the transgenic materials and WT materials, the mature fibers of the two GhMATE27 knockout mutant lines, Ghmate27-L02 and Ghmate27-L16, were observed to be longer than those of the wild type, indicating that knocking out GhMATE27 can improve fiber quality. Targeted metabolomics analysis revealed a significant decrease in catechin content in the petals of the knockout lines, suggesting that GhMATE27 participates in the accumulation of catechins in petals. Figure 5 As shown.

[0119] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0120] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0121] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of GhMATE27 in regulating upland cotton fiber quality and drought resistance of cotton plants, characterized by, The nucleotide sequence of GhMATE27 is shown in SEQ ID NO.

1. Inhibiting the expression of GhMATE27 improves the fiber quality of the upland cotton and enhances the drought resistance of the cotton plant. The improvement in fiber quality is manifested in increasing fiber length.

2. The application according to claim 1, characterized in that, The expression of GhMATE27 can be suppressed by either virus-induced gene silencing or CRISPR-Cas9.

3. The application according to claim 2, characterized in that, The method for virus-induced gene silencing involves amplifying the GhMATE27 fragment using the primers shown in SEQ ID NO.2-3, inserting the GhMATE27 fragment into the pTRV2 vector to obtain the VIGS vector, transforming the VIGS vector with Agrobacterium tumefaciens, and then infecting it with the virus to silence GhMATE27.

4. The application according to claim 2, characterized in that, The CRISPR-Cas9 method involves assembling the sgRNA sequence shown in SEQ ID NO. 15-16 into the vector pYLCRISPR / Cas9P35S-N to obtain the CRISPR-Cas9 vector, and then transforming the CRISPR-Cas9 vector with Agrobacterium to knock out GhMATE27.

5. The application according to claim 1, characterized in that, The upland cotton mentioned is Upland Cotton TM-1.

6. A method for improving the quality of upland cotton fibers, characterized in that, The sgRNA sequence shown in SEQ ID NO.15-16 was assembled into the vector pYLCRISPR / Cas9P35S-N to obtain the CRISPR-Cas9 vector. The CRISPR-Cas9 vector was transformed with Agrobacterium to knock out GhMATE27 as described in claim 1, thereby improving the quality of upland cotton fiber. The improvement in fiber quality is manifested in increasing fiber length.

7. The method according to claim 6, characterized in that, The upland cotton mentioned is Upland Cotton TM-1.

8. A method for improving the drought resistance of cotton plants, characterized in that, The GhMATE27 fragment was amplified using the primers shown in SEQ ID NO.2-3. The GhMATE27 fragment was inserted into the pTRV2 vector to obtain the VIGS vector. The VIGS vector was transformed with Agrobacterium and then used to infect cotton cells, thereby silencing the GhMATE27 described in claim 1 and improving the drought resistance of the cotton plants.

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