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

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of GhMATE27 in regulation and control of upland cotton fiber quality and drought resistance of cotton plants. The invention provides the effect of the GhMATE27 in regulating and controlling the upland cotton fiber quality and the drought resistance of the cotton plant, the drought resistance of the cotton plant is improved by silencing the GhMATE27, and the upland cotton fiber quality is improved by knocking out the GhMATE27.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to the application of GhMATE27 in regulating upland cotton fiber quality and cotton plant drought resistance. Background Art

[0002] Cotton is one of the most important cash crops in the world and the largest source of natural fiber. As my country's main cash crop and textile industry raw material, cotton has formed a huge industrial chain. According to statistics, my country's average annual production and consumption of cotton account for about 21% and 26% of the global total, respectively. The cotton production capacity is seriously insufficient, so that my country has a gap of about 2 million tons in cotton consumption every year. At the same time, the cotton fiber produced in my country is of low quality, high-quality cotton fiber is in serious shortage, low-quality cotton fiber is significantly in excess, and most of the high-end raw cotton market relies on imports. The serious shortage of medium and high-end raw cotton has brought huge challenges to the cotton industry. In addition, my country's main cotton planting areas are the Yangtze River production area, the Yellow River production area and the Xinjiang production area. Among them, the Xinjiang production area is the main cotton production area in my country. In 2023, the output of the Xinjiang production area accounted for about 91% of the total national output. The Xinjiang region has been dry and rainy all year round. The drought caused by water shortage and high temperature has become an important factor restricting local cotton production. Therefore, in order to better adapt to production and living needs, improving cotton quality and drought resistance has become the focus of cotton breeding in my country.

[0003] Flavonoids generally refer to a series of compounds formed by two benzene rings with phenolic hydroxyl groups connected to each other through three central carbon atoms. In terms of chemical structure, flavonoids have three rings, with C6-C3-C6 as their basic skeleton. Based on the degree of oxidation of the central heterocyclic ring, flavonoids are generally divided into seven subclasses, including flavones, isoflavones, flavonols, flavanones, flavanols, chalcones and anthocyanins. In plants, flavonoids are usually found in organs such as flowers, leaves and seeds. At the same time, they usually accumulate in plant cell vacuoles in the form of glycosides. In plants, flavonoids participate in many life activities such as plant growth, development and maturation, and play a vital role. It can 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. At the same time, flavonoids can also help plants resist various biological and abiotic stresses.

[0004] As an important product in cotton production, the yield and quality of cotton fiber directly determine the output value and benefits of cotton production. Therefore, improving the quality of cotton fiber has always been one of the main goals of cotton breeding. Although there are many studies on cotton fiber quality, there are not many studies that associate flavonoids with cotton fiber quality. Studies have found that exogenous application of high concentrations of naringenin and dihydrokaempferol can significantly inhibit the growth and development of cotton fibers, especially elongation, resulting in reduced fiber quality. When the F3H-RNAi fragment was introduced into brown cotton T586, it was also found that the accumulation of naringenin inhibited fiber elongation. Another study found that the flavonoid Rutin has a significant promoting effect on the initiation of cotton fibers, while Naringenin Naringenin inhibits the elongation of cotton fibers in the late fiber development stage. Therefore, the effects of different types of flavonoids on cotton fiber quality at different fiber development stages may be opposite, which requires more systematic and comprehensive research. At present, there are very limited reports on the effects of flavonoids on cotton drought resistance, and further in-depth research is needed. Therefore, improving cotton fiber quality and drought resistance by changing the flavonoid content is a very promising way. However, the molecular mechanism regulating fiber quality and drought resistance of upland cotton has not yet been elucidated. In breeding practice, the available genetic resources for regulating fiber quality and drought resistance of upland cotton are very scarce, making the role of genetic resources in regulating fiber quality and drought resistance of upland cotton unclear. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides the use of GhMATE27 in regulating the fiber quality of upland cotton and the drought resistance of cotton plants.

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

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

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

[0009] Preferably, the method of inhibiting the expression of GhMATE27 includes any one of virus-induced gene silencing and CRISPR-Cas9.

[0010] Preferably, the method for virus-induced gene silencing is to use the primers shown in SEQ ID NO.2-3 to amplify the GhMATE27 fragment, insert the GhMATE27 fragment into the pTRV2 vector to obtain the VIGS vector, transform the VIGS vector with Agrobacterium and then infect it with the virus to silence GhMATE27.

[0011] Preferably, the CRISPR-Cas9 method is to assemble the sgRNA sequence shown in SEQ ID NO.15-16 into the vector pYLCRISPR / Cas9P35S-N to obtain a CRISPR-Cas9 vector, and transform the CRISPR-Cas9 vector by Agrobacterium to knock out GhMATE27.

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

[0013] A method for improving the fiber quality of upland cotton, comprising assembling an sgRNA sequence as shown in SEQ ID NO.15-16 into a vector pYLCRISPR / Cas9P35S-N to obtain a CRISPR-Cas9 vector, and performing Agrobacterium transformation on the CRISPR-Cas9 vector to knock out the GhMATE27, thereby improving the fiber quality of upland cotton.

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

[0015] A method for improving the drought resistance of the cotton plant, comprising: using the primers shown in SEQ ID NO.2-3 to amplify the GhMATE27 fragment, inserting the GhMATE27 fragment into the pTRV2 vector to obtain the VIGS vector, and subjecting the VIGS vector to Agrobacterium transformation and virus infection to silence the GhMATE27, thereby improving the drought resistance of the cotton plant.

[0016] Compared with the prior art, the present invention is beneficial in that:

[0017] The present invention proposes the role of GhMATE27 in regulating the fiber quality of upland cotton and the drought resistance of cotton plants, improves the drought resistance of the cotton plants by silencing GhMATE27, and improves the fiber quality of upland cotton by knocking out GhMATE27. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the accumulation spectrum of flavonoids in different tissues of 383 upland cotton materials.

[0019] Figure 2 Metabolite-based genome-wide association analysis.

[0020] Figure 3 For protoplast subcellular localization experiments.

[0021] Figure 4 This is a drought tolerance experiment under VIGS treatment.

[0022] Figure 5It is a phenotypic verification experiment for transgenic knockout materials. DETAILED DESCRIPTION

[0023] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.

[0024] Example 1

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

[0026] To analyze the effects of flavonoids in upland cotton petals on cotton fiber quality and cotton 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 flowering day, which was recorded as 0 DPA, and were quick-frozen with liquid nitrogen and then stored in a -80°C refrigerator.

[0027] Targeted metabolomics assays

[0028] The petals at 0 DPA of flowering were taken as samples, with each material weighing 0.3 g to 0.5 g fresh weight, and two biological replicates were set. Then, through targeted metabolomics, the content of 16 flavonoid compounds in cotton petals was detected by liquid chromatography-mass spectrometry.

[0029] (1) Sample preparation: fresh materials were placed in 2 mL EP tubes, immediately frozen in liquid nitrogen, and stored in a -80°C ultra-low temperature freezer. The day before sample preparation, freeze-dried the corresponding tissue material for 16 h, accurately weighed 0.100 g with a 1 / 10,000 balance, crushed into powder with a ball mill, added with extract at a ratio of 0.1 g / mL, vortexed for 15 min, centrifuged at 4°C 15000 rpm in a frozen centrifuge for 30 min, filtered with a 0.22 μm filter membrane, and 150 μL to 200 μL were placed in a vial for testing using a liquid chromatography-mass spectrometry instrument QTrap4000. The extract was 80% methanol water by volume, containing chrysin as an internal standard.

[0030] (2) On-machine detection: This experimental instrument uses an ESI ion source. The retention time RT / s of the detected Q1 parent ion and Q3 fragment ion is compared with the retention time of the standard. The detected ions are qualitatively analyzed. If the retention time is the same, it can be determined to be the same compound. The peak area is 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 with a volume fraction of 0.05% formic acid aqueous solution and phase B with a volume fraction of 0.05% formic acid acetonitrile. The experiment uses flavonoid standards as external standards, sets a concentration gradient, and makes a standard curve. At the same time, chrysin in the extract is used as an internal standard to eliminate machine noise and reduce instrument errors.

[0031] Results: A total of 16 flavonoids from 7 categories were detected in the petals. It was found that the content of flavonols was high, while the content of other flavonoids was low, indicating that the metabolism of flavonols in the petals was relatively active. At the same time, it was found that the accumulation of flavonols had obvious group specificity, such as Figure 1 shown.

[0032] Example 2

[0033] Discovering the key genes that regulate flavonoid content in cotton petals

[0034] (1) Descriptive statistical analysis: By sorting out the data distribution characteristics of all detected flavonoid compounds 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 conformed to the normal or approximately normal distribution, which was suitable for subsequent GWAS analysis.

[0035] (2) Metabolite-based whole-genome association analysis: A large amount of data was obtained by conducting targeted metabolomics determination of flavonoid metabolites in petals of 383 natural population materials of upland cotton, and then mGWAS was performed on it together with the resequencing genome data. We used 1,076,652 high-quality SNP data to ensure that the minor allele frequency was >0.05 and the missing rate was <0.2. At the same time, we used a mixed linear model to perform mGWAS and identified 3778 SNP sites that were highly correlated with flavonoids above the medium significance threshold. At the same time, we drew a Manhattan plot of the petal catechin content as a target trait, and simultaneously performed LD block analysis and haplotype analysis, such as Figure 2 shown.

[0036] (3) Determination of candidate genes: Finally, we identified a total of 50 quantitative trait loci (QTLs), including 235 candidate genes, which are related to the flavonoid content in specific tissues. Among them, the gene involved in the extrusion transporter of multidrugs and toxic compounds was designated as GhMATE27; we then further studied the effects of significant SNPs on the function of each gene and found that a SNP on GhMATE27 caused premature translation termination, which had a significant impact on protein structure and function. Therefore, GhMATE27 was selected as a candidate gene for subsequent functional verification.

[0037] Example 3

[0038] Functional verification of GhMATE27

[0039] The gene number of GhMATE27 is Gh A06G036100, and the 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, recorded as SEQ ID NO.1.

[0070] 1. Protoplast subcellular localization

[0071] By combining the GFP tag with the GhMATE27 protein and the localization of the γTIP-RFP tag, it was found that the protein was localized on the vacuole membrane, e.g. Figure 3 shown.

[0072] VIGS Experiment

[0073] 1. VIGS vector construction

[0074] (1) Cloning the target gene fragment: Use PCR technology to amplify the target gene fragment. The target gene is GhMATE27, and a sequence with a coding region length of about 300-500 bp is selected.

[0075] Amplification primers are as follows:

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

[0077] R: AAAGGTACCATCACTGTCTGAATAGCTGTTCC, recorded 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, which was then sequenced to ensure 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. Virus 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 mixed solution 1, and the Agrobacterium solution containing the VIGS vector was resuspended in VIGS buffer to obtain mixed solution 2. Mixed solution 1 and mixed solution 2 were mixed in equal proportions and activated at 28°C for 3 h to obtain a virus suspension, wherein the VIGS buffer contained 10 mmol / L MgCl2, 10 mmol / L MES and 200 μmol / L AS, and had a pH of 5.6.

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

[0085] The experiment used plants transformed with TRV:CLA1 as a positive control, CLA1 is GhCLA1, a key enzyme for chloroplast synthesis. TRV:00 (empty vector) plants were used as negative controls. Three parallel experiments were set up for each experimental group.

[0086] The expression of GhMATE27 was reduced by virus-induced gene silencing (VIGS), and it was observed that the material after gene silencing experienced severe wilt under drought conditions and the contents of H2O2 and malondialdehyde (MDA) increased significantly compared with the control group, indicating that the expression of GhMATE27 can indeed improve the drought resistance of cotton plants. Figure 4 shown.

[0087] 3. Transgenic Experiment

[0088] 1. Construct CRISPR-Cas9 vector according to Ma et al., 2015, Molecular plant. The specific method is as follows:

[0089] (1) Synthesize sgRNA

[0090] Using pYLgRNA-AtU3b as a template, primer UF and sgRNA-xR were used for PCR reaction, and the obtained product was recorded as the first round PCR product A. In sgRNA-xR, x is 1 or 2.

[0091] Using pYLgRNA-AtU6-29 as a template and primers sgRNA-xF and gR-R, a PCR reaction was performed, and the obtained product was recorded as the first-round PCR product B. In sgRNA-xF, x is 1 or 2.

[0092] The first-round PCR product A and the first-round PCR product B were used as templates, and the primer pair Pps-GGL and Pgs-GG2R, and the primer pair Pps-GG2F and Pgs-GGR were used to clone the complete sgRNA, respectively.

[0093] The nucleotide sequence of 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 Escherichia coli, positive clones were screened by resistance, and plasmid extraction and sequencing were performed for verification. The recombinant vector, namely the CRISPR-Cas9 vector, was obtained by correct sequencing.

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

[0113] (1) Creation of genetically modified 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 sequenced for identification. The target site sequence mutation was the mutant material, and the mutant materials Ghmate27-L02 and Ghmate27-L16 were obtained.

[0117] (3) Phenotypic measurement of mutants

[0118] The creation of transgenic knockout materials was completed through the CRISPR-Cas9 system. First, there was no significant difference in the growth phenotype between the transgenic materials and the WT. At the same time, in the three parallel experiments set up to compare the fiber length of the transgenic materials and the WT, it was observed that the mature fibers in the two knockout mutant lines of GhMATE27, Ghmate27-L02 and Ghmate27-L16, were longer than those in the wild type, indicating that knocking out GhMATE27 can improve the quality of the fiber. Through targeted metabolomics determination, it was found that the catechin content in the petals of the knockout lines was significantly reduced, indicating that GhMATE27 is involved in the accumulation of catechins in the petals, such as Figure 5 shown.

[0119] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0120] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0121] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Application of GhMATE27 in regulating upland cotton fiber quality and cotton plant drought resistance, characterized in that: The nucleotide sequence of GhMATE27 is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The expression of GhMATE27 is inhibited to improve the fiber quality of upland cotton.

3. The use according to claim 1, characterized in that: The expression of GhMATE27 is inhibited to improve the drought resistance of the cotton plant.

4. The use according to claim 1, characterized in that: The methods for inhibiting the expression of GhMATE27 include any one of virus-induced gene silencing and CRISPR-Cas9.

5. The use according to claim 4, characterized in that: The method for virus-induced gene silencing is to use the primers shown in SEQ ID NO.2-3 to amplify the GhMATE27 fragment, insert the GhMATE27 fragment into the pTRV2 vector to obtain the VIGS vector, and then perform Agrobacterium transformation on the VIGS vector and then perform virus infection to silence GhMATE27.

6. The use according to claim 4, characterized in that: The CRISPR-Cas9 method is to assemble the sgRNA sequence shown in SEQ ID NO.15-16 into the vector pYLCRISPR / Cas9P35S-N to obtain a CRISPR-Cas9 vector, and transform the CRISPR-Cas9 vector with Agrobacterium to knock out GhMATE27.

7. The use according to claim 1, characterized in that: The upland cotton is upland cotton TM-1.

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

9. The method according to claim 8, characterized in that The upland cotton is upland cotton TM-1.

10. A method for improving drought resistance of cotton plants, characterized in that: The GhMATE27 fragment is amplified using the primers shown in SEQ ID NO.2-3, and the GhMATE27 fragment is inserted into the pTRV2 vector to obtain the VIGS vector. The VIGS vector is transformed with Agrobacterium and then infected with cotton cells, so that the GhMATE27 described in claim 1 is silenced, thereby improving the drought resistance of the cotton plant.

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