Application of cotton GhWRKY46 gene in regulation and control of cotton fiber quality

Abstract

The invention belongs to the technical field of plant gene editing, and particularly relates to application of a cotton GhWRKY46 gene in regulation and control of cotton fiber quality. The invention provides an application of a GhWRKY46 gene in regulation and control of cotton fiber quality. An amino acid sequence coded by the GhWRKY46 gene is as shown in SEQ ID NO: 1. According to the method, the endogenous GhWRKY46 gene of upland cotton is precisely edited, so that the fiber length of the obtained gene edited cotton is increased, the thickness of a secondary wall is reduced, the micronaire value is reduced, the specific breaking strength is improved, the cotton fiber quality is remarkably improved, and a cotton excellent germplasm resource with the fiber length and strength improved is obtained. According to the gene provided by the invention, the comprehensive quality of cotton fibers is remarkably superior to that of wild cotton fibers, and the gene has important agricultural application value.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant genetic engineering, and particularly relates to application of a cotton GhWRKY46 gene in regulating cotton fiber quality. BACKGROUND

[0002] Cotton is an important economic crop, and its fiber is an important raw material for the textile industry in the world. Meanwhile, cotton is also an important source of oil, feed and other industrial raw materials. The fiber quality (such as length, strength, micronaire) of cotton is a key factor determining the economic value of cotton.

[0003] With the development of society, people's requirements for the quality of cotton textiles are continuously improved, therefore, cotton spinning enterprises pay more and more attention to using longer, stronger, thinner and more uniform cotton fibers as textile raw materials for producing fine and exquisite cotton yarns and cotton textiles. However, the deficiency of cotton fiber quality restricts the development of the textile industry, and improving the quality of cotton fiber helps to break the bottleneck restricting the economic development of cotton, which puts forward new requirements for cotton molecular breeding.

[0004] Therefore, exploring the transcription factors and their coding genes related to the quality of cotton fiber to improve the quality of cotton fiber and promote the development of the textile industry has become a problem to be solved in the field. SUMMARY

[0005] In order to solve the problem of lack of coding genes closely related to the quality of cotton fiber in the prior art, the application provides application of a cotton GhWRKY46 gene in regulating cotton fiber quality, and specifically comprises the following technical solutions.

[0006] The application provides application of a GhWRKY46 gene in regulating cotton fiber quality, and the amino acid sequence coded by the GhWRKY46 gene is shown in SEQ ID NO: 1.

[0007] Preferably, the nucleotide sequence of the GhWRKY46 gene is shown in SEQ ID NO: 2.

[0008] Preferably, the regulation of cotton fiber quality comprises knocking out or reducing the expression of the GhWRKY46 gene in cotton to improve the quality of cotton fiber.

[0009] Preferably, the cotton fiber quality comprises one or more of the fiber length, fiber elongation rate, breaking specific strength and micronaire value of cotton.

[0010] The application also provides application of a biological material for negatively regulating a GhWRKY46 gene in regulating cotton fiber quality.

[0011] The application also provides a biological material for negatively regulating the expression of a cotton GhWRKY46 gene, including a biological material for knocking out or inhibiting the expression of a cotton GhWRKY46 gene.

[0012] The biological material includes one or more of a transformant of a GhWRKY46 gene, an sgRNA, a primer pair of the sgRNA, a recombinant vector containing the sgRNA, and a recombinant microorganism containing the sgRNA.

[0013] Preferably, the transformant of the GhWRKY46 gene includes transformant ko4 and / or transformant ko5.

[0014] The transformant ko4 is that CCA is deleted at positions 1075-1077 and ATGGGCAAC is deleted at positions 1079-1087 of the GhWRKY46 gene.

[0015] The transformant ko5 is that a G base is inserted at position 1078 of the GhWRKY46 gene.

[0016] Preferably, the sequence of the sgRNA is shown as SEQ ID NO: 7.

[0017] Preferably, the primer pair of the sgRNA is shown as SEQ ID NO: 9 and SEQ ID NO: 10.

[0018] The application also provides a breeding method for improving the quality of cotton fibers, in which the expression of a GhWRKY46 gene in the cotton is knocked out or reduced, and a cotton plant with high-quality cotton fibers is obtained.

[0019] The application has the following beneficial effects:

[0020] The application provides an application of a GhWRKY46 gene in regulating the quality of cotton fibers, in which the amino acid sequence encoded by the GhWRKY46 gene is shown as SEQ ID NO: 1. Through precise editing of an endogenous GhWRKY46 gene in Gossypium hirsutum, the gene editing cotton has increased fiber length, thinned secondary wall thickness, reduced micronaire value, and improved breaking specific strength, and the quality of the cotton fibers is significantly improved, and a cotton excellent germplasm resource with improved fiber length and strength is obtained. The gene provided by the application makes the comprehensive quality of the cotton fibers significantly better than that of the wild type, and has important agricultural application value. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed in the embodiments will be briefly introduced below.

[0022] Figure 1Expression pattern of GhWRKY46 in different tissues of wild type Gossypium hirsutum YZ-1

[0023] Wherein, 5-25DF is fiber at 5-25 days after flowering, 0-10DO is ovule at 0-10 days after flowering, Root is root, Young stem is young stem, Old stem is old stem; Leaf is leaf; Petal is petal, Pistil is pistil, Stamens is stamen;

[0024] Figure 2 Structure diagram of gene editing vector pRGEB32-GhWRKY46;

[0025] The gene editing vector pRGEB32-GhWRKY46 contains sgRNA expression frame and Cas9 gene;

[0026] Figure 3 Identification results of gene editing plants;

[0027] Wherein, A figure is identification results of homozygous editing line with or without exogenous DNA insertion; Figure 2 In A, M: DL2000; P: positive control, using pRGEB32 plasmid as template; N: negative control, using wild type cotton leaf gDNA as template; W: using water as amplification template; GUS+, GUS- respectively represent GUS staining positive and negative; 1, 2, 5, 6 use cotton leaf gDNA of ko4 offspring of homozygous mutant as template; 3, 4, 7, 8 use cotton leaf gDNA of ko5 offspring of homozygous editing line as template;

[0028] B figure is identification of editing site of offspring separation line of GhWRKY46 gene editing line;

[0029] Figure 4 Analysis results of mature fiber secondary wall thickness of wild type and gene editing plants;

[0030] Wherein, A figure is paraffin section of mature fiber of wild type and GhWRKY46 gene editing line, scale bar 20 μm;

[0031] B figure is statistical results of fiber cell wall thickness, error bar represents standard deviation of 50 fiber cell wall thickness statistics; ** represents P<0.01;

[0032] Figure 5 Analysis results of mature fiber length of wild type and gene editing plants;

[0033] Figure A shows the mature fibers of wild type and GhWRKY46 gene edited lines, and the scale bar is 1 cm; Figure B shows the mature fiber length statistics of wild type and GhWRKY46 gene edited lines, and the error bar indicates the standard deviation of 30 mature fiber length statistics, and ** indicates P<0.01. DETAILED DESCRIPTION

[0034] The application provides an application of a GhWRKY46 gene in regulating cotton fiber quality, wherein the amino acid sequence encoded by the GhWRKY46 gene is shown as SEQ ID NO: 1.

[0035] GhWRKY46 (SEQ ID NO: 1): MEKTMGWEQNTLLNELAQGRDFTNMLRKHLHPSSSPETRQVLLDKILCSYDKALSLLNCSRFMVETKPRVRTLGSPENDASDNKDMFKKRKTSSGWSEQIRVCSAMSLEGPLDDGYCWRKYGQKDILGSNFPRAYYRCTHRYSQGCLAGKQVQRSDEDPTIFEVKYRGRHACNQVPHLVATPKEKGNHYREKQQVEEKQKQSKEMLLSFETGLKVKTEDLDNREDIFPSFSFPIESEEVQNGLLLNSLMKNMSPAFVSPATSESNYFSVSAFHMGNFDFGQNVQTSESELTEIISAPASVTNSPIVDLDISSLEKLELDQSFPYDNPEFFTNFLQ.

[0036] As an embodiment, the nucleotide sequence of the GhWRKY46 gene is shown as SEQ ID NO: 2.

[0037] GhWRKY46 gene (SEQ ID NO: 2): 5'-ATGGAAAAAACGATGGGTTGGGAGCAAAACACTCTTTTGAATGAGCTAGCACAGGGGAGAGACTTCACTAATATGCTAAGGAAACACCTCCATCCATCTTCATCCCCCGAAACACGCCAAGTCTTGCTTGACAAAATACTGTGCTCTTATGACAAAGCTCTTTCGTTGCTGAATTGTAGTCGTTTTATGGTTGAGACGAAACCCAGGGTTCGCACGTTGGGATCCCCTGAAAATGACGCCTCTGATAACAAGGATATGTTTAAAAAGAGGAAAACATCTTCAGGGTGGAGTGAGCAAATAAGGGTTTGCTCGGCGATGTCATTAGAGGGGCCTCTTGATGATGGATATTGCTGGCGAAAATATGGGCAGAAAGATATTCTTGGATCCAATTTTCCAAGGGCATATTATCGATGCACTCATCGTTACTCGCAAGGCTGCTTAGCCGGTAAGCAAGTTCAAAGATCAGACGAGGACCCAACAATATTCGAGGTGAAATATCGTGGAAGACATGCCTGTAACCAAGTCCCTCACTTGGTTGCTACGCCCAAAGAGAAAGGCAATCATTATCGGGAAAAGCAGCAAGTTGAAGAGAAACAAAAGCAATCCAAGGAAATGTTGTTGAGTTTTGAAACAGGGCTTAAGGTTAAAACAGAGGACTTGGACAACAGAGAGGACATATTTCCTTCATTTTCTTTCCCCATTGAGTCAGAGGAAGTTCAAAATGGGTTATTGCTTAATTCCCTAATGAAAAACATGTCTCCGGCATTTGTGTCACCAGCTACATCTGAATCCAACTATTTTTCAGTGTCAGCGTTCCACATGGGCAACTTTGATTTCGGCCAAAATGTGCAGACTTCGGAATCTGAACTTACAGAAATAATCTCTGCACCAGCTTCAGTTACTAATTCACCCATTGTCGACCTCGATATTTCATCACTCGAGAAGCTGGAGTTAGATCAAAGCTTCCCATATGATAATCCTGAGTTCTTCACTAATTTTTTACAGTAA-3’;

[0038] The GhWRKY46 gene is a member of the WRKY transcription factor family. The protein encoded by the gene contains a WRKY domain composed of about 60 amino acids, a highly conserved WRKYGQK core motif and a zinc finger motif, and can be involved in the regulation of the transition from fiber elongation to secondary wall development. The present application uses CRISPR / Cas9 technology to achieve efficient knockout of the cotton GhWRKY46 gene, prolongs the fiber cell elongation time, shortens the secondary wall synthesis time, and thus obtains longer and thinner secondary wall fibers, so that the mature fibers have excellent quality traits of being long, soft and low micronaire value.

[0039] As an embodiment, the method for regulating cotton fiber quality comprises knocking out or reducing the expression of the GhWRKY46 gene in cotton to improve the cotton fiber quality. As an embodiment, the cotton fiber quality comprises one or more of the fiber length, fiber elongation rate, breaking specific strength and micronaire value of the cotton.

[0040] The present application also provides a biological material for negatively regulating the GhWRKY46 gene for use in regulating the cotton fiber quality. As an embodiment, the GhWRKY46 gene is as described above and will not be repeated here.

[0041] The present application also provides a biological material for negatively regulating the expression of the cotton GhWRKY46 gene, which comprises a biological material for knocking out or inhibiting the expression of the cotton GhWRKY46 gene. The biological material comprises one or more of a transformant of the GhWRKY46 gene, an sgRNA, a primer pair of the sgRNA, a recombinant vector containing the sgRNA, and a recombinant microorganism containing the sgRNA.

[0042] As an embodiment, the transformant of the GhWRKY46 gene comprises a transformant ko4 and a transformant ko5. The transformant ko4 is a deletion of CCA at positions 1075-1077 and a deletion of ATGGGCAAC at positions 1079-1087 of the GhWRKY46 gene. The transformant ko5 is an insertion of a G base at position 1078 of the GhWRKY46 gene. As an embodiment, the transformant ko4 and the transformant ko5 correspond to the ko4 strain and the ko5 strain described in embodiments 4-7, respectively. The ko4 strain has a deletion of CCA at positions 1075-1077 and a deletion of ATGGGCAAC at positions 1079-1087 of the GhWRKY46 gene, which affects the normal transcription of the gene and thus inhibits the expression of the GhWRKY46 gene. The ko5 strain has an insertion of a G base at position 1078 of the GhWRKY46 gene, which causes premature termination of translation of the GhWRKY46 gene.

[0043] As an implementation form, the sequence of the sgRNA is shown in SEQ ID NO: 7. As an implementation form, the sgRNA targets the 3rd exon of the GhWRKY46 gene, and the PAM sequence of the sgRNA is NGG.

[0044] As an implementation form, the primer pair of the sgRNA is shown in SEQ ID NO: 9 and SEQ ID NO: 10. The SEQ ID NO: 9 contains the forward sequence of the sgRNA and a connecting sequence; the SEQ ID NO: 10 contains the reverse complementary sequence of the sgRNA and a connecting sequence.

[0045] As an implementation form, the recombinant vector containing the sgRNA includes a backbone vector and the sgRNA. As an implementation form, the backbone vector includes a plasmid vector; as another implementation form, the plasmid vector includes a pRGEB32 vector. As an implementation form, the recombinant vector containing the sgRNA of the present application is a CRISPR / Cas9 gene editing vector pRGEB32, which can be used for improving the fiber quality of cotton, and the structure of the T-DNA region thereof includes: the left border of the T-DNA - the terminator NOS of the Agrobacterium crown gall synthetic enzyme gene - the fusion gene expression cassette of the cauliflower mosaic virus CaMV35S promoter with the reporter gene GUS (β-glucuronidase gene) and the marker gene NPTII (neomycin phosphotransferase gene) in series - the promoter GhU6_7-tRNA fusion sgRNA-gRNA scaffold - the rice ubiquitin Ubi promoter OsUbiPromoter (Cas9 protein driving promoter) - 3xFLAG tag - simian virus 40 nuclear localization peptide signal SV40NLS - Cas9 protein coding sequence (SEQ ID NO. 12) - NLS sequence fused with nucleoplasmin - NOS - the right border of the T-DNA, and the structure thereof is shown in Figure 1 .

[0046] As an implementation form, the recombinant microorganism containing the sgRNA includes a basic microorganism and the sgRNA. As an implementation form, the basic microorganism includes bacteria; as another implementation form, the bacteria include Agrobacterium and Escherichia coli. In a specific implementation form, the Agrobacterium is Agrobacterium LBA4404; the Escherichia coli is E. coli DH5α.

[0047] The application also provides a breeding method for improving the fiber quality of cotton, comprising the following steps: knocking out or reducing the expression of the GhWRKY46 gene in the cotton to obtain a cotton plant with high fiber quality. As an embodiment, the breeding method uses biological materials to knock out or reduce the expression of the GhWRKY46 gene, and the biological materials are the same as described above, which will not be repeated here.

[0048] As an embodiment, the breeding method for improving the fiber quality of cotton comprises the following steps: introducing a gene editing vector containing the sgRNA into Agrobacterium LBA4404 competent cells by electroporation, using Agrobacterium-mediated method to infiltrate cotton embryogenic callus, obtaining resistant buds through kanamycin screening, transplanting the resistant buds to a greenhouse after inducing rooting, verifying the deletion mutation of the GhWRKY46 gene through PCR amplification and Hi-TOM sequencing, screening homozygous mutant lines without exogenous gene insertion through GUS staining and PCR amplification, and obtaining a cotton variety with improved fiber quality.

[0049] In order to further illustrate the application, the application of the cotton GhWRKY46 gene in regulating the fiber quality of cotton is described in detail below in combination with the drawings and examples, but they should not be understood as limiting the scope of protection of the application.

[0050] Unless otherwise specified, the reagents and drugs in the application are ordinary commercially available. Unless otherwise specified, the materials and methods are referred to from “Li Fuguang, Yuan Youlu. Cotton Molecular Breeding. Beijing: China Agricultural University Press, 2013.05”.

[0051] Example 1 Expression pattern analysis of GhWRKY46 gene

[0052] 1. Sampling

[0053] The wild type Gossypium hirsutum YZ-1 was used as an experimental sample, and the cotton fiber and ovule tissue samples were collected at 0DPA, 5DPA, 10DPA, 15DPA, 20DPA and 25DPA of the wild type Gossypium hirsutum YZ-1 cotton boll development, and the cotton second leaf, root, stem, leaf, flower and other tissue samples were collected. The above collected tissue samples were quickly frozen with liquid nitrogen and stored in a-80℃ refrigerator.

[0054] 2. RNA extraction

[0055] The RNA in the above tissue samples was extracted using an RNA extraction kit (Tiangen Plant RNA Quick Kit DP452), and the specific steps were as follows:

[0056] Each 100 mg of fiber tissue samples were mixed at each DPA, ground into powder in liquid nitrogen, and quickly transferred to an RNase-free centrifuge tube containing 600 μL of lysis solution RLT (RNA extraction kit components). The sample was fully lysed by vortexing. The lysis solution was filtered through a column filter provided with the kit into a new centrifuge tube, and 0.5 times the volume of anhydrous ethanol was added and mixed well by inverting. The mixture was transferred to the adsorption column, which was centrifuged at 12,000 rpm for 1 min at 4°C, and the filtrate was discarded. The adsorption column was washed with 700 μL of rinse solution RW1 (provided in the kit), centrifuged at 12,000 rpm for 1 min at 4°C, and the filtrate was discarded. 500 μL of rinse solution RW2 (anhydrous ethanol was added before use according to the instructions) was added to the adsorption column, which was centrifuged at 12,000 rpm for 1 min at 4°C, and the filtrate was discarded. The operation was repeated once. The empty column was centrifuged at 12,000 rpm for 2 min at 4°C to remove residual liquid. The adsorption column was placed in a new RNase-free centrifuge tube, and 30-50 μL of RNase-free water was added to the center of the column membrane, which was allowed to stand at room temperature for 2 min, and then centrifuged at 12,000 rpm for 1 min at 4°C to collect the RNA solution. The spectrophotometer was used to determine the concentration and purity of the RNA, and the OD 260 / OD 280 ratio was required to be between 1.8 and 2.0, and the qualified RNA was stored at -80°C for later use.

[0057] 3. Reverse transcription

[0058] The reaction system was configured according to the instructions of the All-in-one one-step reverse transcription kit (GenStar Star Script Pro All-in-one RT Mix With gDNA Remover, item number: A240-10), and the following operations were performed:

[0059] 1 μg of total RNA template, 1 μL of Reaction Buffer, 4 μL of All-in-one RT Enzyme Mix, and RNase-free water were added to make up to 20 μL. The reaction program was set as follows: 37°C for 10 min (to eliminate genomic DNA), 55°C for 15 min (reverse transcription), and 85°C for 5 min (enzyme inactivation). After the reaction was completed, the cDNA was stored at -20°C.

[0060] 4. Real-time fluorescent quantitative PCR

[0061] The reaction system (20 μL) was as follows: 10 μL of 2×SYBR Green PCR Master Mix, 1 μL of Gh WRKY46 upstream primer (SEQ ID NO: 3) (5 μM), 1 μL of Gh WRKY46 downstream primer (SEQ ID NO: 4) (5 μM), 3 μL of cDNA template, and ddH2O 5 μL.

[0062] Reaction program: 95℃ pre-denaturation for 30s; 95℃ denaturation for 5s, 60℃ annealing for 30s, 40 cycles.

[0063] RT-GhWRKY46-F (SEQ ID NO:3): 5'-TCTCCGGCATTTGTGTCACCAG-3';

[0064] RT-GhWRKY46-R (SEQ ID NO:4): 5'-CGAAGTCTGCACATTTTGGCC-3'.

[0065] Actin was used as the internal reference gene. The primers for the Actin internal reference gene are as described in SEQ ID NO:5 and SEQ ID NO:6. The reaction system and procedure were the same as above. Each sample was replicated in triplicate, using 2... -ΔΔCt The method calculates the relative expression level of genes.

[0066] Actin primer F (SEQ ID NO:5): 5'-TTGCAGACCGTATGAGCAAG-3';

[0067] Actin primer R (SEQ ID NO:6): 5'-ATCCTCCGATCCAGACACTG-3'.

[0068] 5. Results Analysis

[0069] like Figure 1 As shown in the qRT-PCR analysis, the expression level of the GhWRKY46 gene was low in the early stage of fiber development (0DP A–15DPA), and increased significantly after 15DPA, reaching a peak at 20–25DPA, exhibiting a late-stage dominant expression characteristic. This verifies the late-stage dominant expression characteristic of GhWRKY46 in fiber development.

[0070] Example 2: Construction of the GhWRKY46 gene CRISPR / Cas9 gene editing vector

[0071] 1. GhWRKY46 gene target design

[0072] Based on the GhWRKY46 gene sequence shown in SEQ ID NO:2, sgRNA targets were designed using CRISPR-P2.0 software. The designed sgRNA is shown in SEQ ID NO:7, and the sgRNA targets exon 3 of the GhWRKY46 gene.

[0073] SgRNA (SEQ ID NO:7): 5'-TCAGTGTCAGCGTTCCACATGGG-3';

[0074] The PAM sequence of the sgRNA is NGG.

[0075] 2. Construction of sgRNA expression cassette vector

[0076] The sgRNA is repeated in series driven by the cotton U6-7 promoter as shown in SEQ ID NO: 8, and the specific steps are as follows:

[0077] GhU6-7 (SEQ ID NO: 8): 5'-ttaatctgatgctccacctgcttttgattttctttattggaagagtctttaagagatatgttaagtagcataacagtttcatcaaaaacaacatttctgttaatcacaacttttctattttcaggataccataacttatacacttttacactagctttataaccaagaaaaacacatttaatggtacacaattttaattttccattatcagcatgagtatacgcaaaacacccaaaaatctttaaatcagaatcgtcagcaggattactaaaccatacttcttatggagtctttttctcaatagcaacgaatagagacggattgatcaaaaacatatagttgacattgctttggcccaaaataactttgataagttgccatttgacaacatacatcgaacattctccatgatcgttctattcattcgttctacaataccttttttaaaatgttcaggttctaaaatgaaaaacaatatgaattgcatgaattgcttatatgtcctatgaattataaaggaatgcggttgaaatattcccatcgatacatacatacatattcgtgaagtatgttccaatataatatcaatattgggatttacgttttataaagcaacattattgattggtaatatacattaattccaaggcaaacccaaatattttaaaatttaacctacaactgtggtaaatcaaacttaatagtaacccgattgtaatgtgaagtcaaatatgaaagtaacattggtttatatatatattttctctaaattctaataatcaagttgggataagtgataaacactgagcttgccacgtgtgttaacctcgttttcatcatgtgccactccaaagacatcaggcctctattcaagctggcatggtcaggacgtggtagcatacttcagggatctggttagaaaatatcccatatcgctaaagaactataacacaggagcgtttatataagcgaaagaagcatcagatgg-3’.

[0078] Primers were designed for sgRNAs, and the primer sequences are shown below:

[0079] sgRNA-F (SEQ ID NO: 9): 5'-attcccggctggtgcaTCAGTGTCAGCGTTCCACATgttttagagctagaaatagcaag-3';

[0080] SEQ ID NO: 9 contains sgRNA forward sequence (first capital sequence) and connecting sequence (first lowercase sequence and second lowercase sequence);

[0081] sgRNA-R (SEQ ID NO: 10): 5'-ctatttctagctctaaaacATGTGGAACGCTGACACTGAtgcaccagccgggaatcgaa-3';

[0082] SEQ ID NO: 10 contains sgRNA reverse complement sequence (first capital sequence) and connecting sequence (first lowercase sequence and second lowercase sequence).

[0083] The substrate is pUC57-gRNA scaffold-tRNA, as shown in SEQ ID NO: 11.

[0084] gRNA scaffold-tRNA (SEQ ID NO: 11): 5'gttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcaacaaagcaccagtggtctagtggtagaatagtaccct gccacggtacagacccgggttcgattcccggctggtgca-3';

[0085] The amplification procedure is as follows: 95℃ pre-denaturation for 5 min, cycle (35 times), 98℃ denaturation for 10 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, and 72℃ for 5 min for termination.

[0086] The amplification system (20 μL) comprises: 2 x Taq Master Mix (Dye Plus) 10 μL (Novozyme P112-03), 1 μL of primer (5 μmol / L) (synthesized by Huada Gene), about 100 ng of template DNA, and ddH2O added to 20 μL.

[0087] Gene editing vector enzyme digestion: linearize the backbone pRGEB32 using specific endonuclease Eco311, the enzyme digestion system (50 μL) is: 3 μg plasmid, 5 μL 10×Green buffer, 3 μL Eco311, add ddH2O to 50 μL.

[0088] Enzyme digestion product and amplification product processing: load the amplification product and 5 μL DNA Marker (1 kb Ladder) on a 1% gel for electrophoresis, 1×TAE buffer, 120V constant voltage treatment for 22 min, observe the band position under the gel imaging system as 200 bp size, cut the gel block containing the target band under the ultraviolet lamp, use the agarose gel recovery kit (Zhuangmeng ZPN202-3) for recovery, the enzyme digestion product does not need to be electrophoresed, and is directly recovered.

[0089] Seamless cloning connection, transformation:

[0090] Mix the target fragment and the linearized vector according to the appropriate proportion into the seamless cloning kit (Gen STAR Seamless Cloning IE161B) reaction system, incubate at 50°C for 20 min, then transform the ligation product into competent cells E. coli (DH5α) by heat shock method, add LB medium and shake culture, then centrifuge and collect the cells and spread on an antibiotic-containing plate, finally verify the positive clone by colony PCR and sequencing screening. The structure of the constructed gene editing vector pRGEB32-GhWRKY46 is shown in Figure 2 .

[0091] The constructed CRISPR / Cas9 vector is introduced into Agrobacterium LBA4404 competent cells by electroporation, mix 100 ml of competent LBA4404 with 4 μg of plasmid, ice bath for 5 min, set the parameters of the electroporation instrument to 2.5 kV / 200 Ω / 25 μF, click to add 1 mL of antibiotic-free YEB medium, shake culture at 28°C for 2 h, spread on a YEB plate containing kanamycin (50 mg / L) and rifampicin (100 mg / L), and screen positive clones. The 3×FLAG-Cas9-nucleoplasmin NLS protein coding sequence is shown in SEQ ID NO: 12.

[0092]

[0093] Example 3 Agrobacterium-mediated genetic transformation of cotton and regeneration of plants

[0094] In this example, the culture medium and its components are shown in Table 1.

[0095] Table 1 Culture medium and its components

[0096]

[0097]

[0098] In this example, the formula of the 100xMSB stock solution is as follows: macroelements (17 g / L KH2PO4, 37 g / L MgSO4·7H2O, 33.224 g / L CaCl2, 2.78 g / L FeSO4·7H2O, 3.732 g / L Na2·EDTA); microelements (0.31 g / L H3BO3, 0.0415 g / L KI, 0.0125 g / L Na2MoO4·2H2O, 0.84505 g / L MnSO4·H2O, 0.43 g / L ZnSO4·7H2O, 0.01225 g / L CuSO4·5H2O, 0.00125 g / L CoCl2·6H2O); organic substances (5 g / L myo-inositol, 0.05 g / L nicotinic acid, 0.05 g / L VB6, 0.5 g / L VB1); agar Gelrite: Sigma, item number: G1910; SH: Schenk & Hildebrandt, 1972. Note: during the experiment, the final concentration of the 100xMSB stock solution in the culture medium is 10 ml / L.

[0099] Inoculation preparation: a single colony is inoculated in 5 mL YEB liquid medium (containing 50 mg / L kanamycin, 100 mg / L rifampicin) and cultured at 28°C for 24 h with shaking until the OD 600 = 1.0, using the hypocotyl transformation method with the genetic background of the wild type YZ-1 upland cotton variety.

[0100] Explant preparation: the YZ-1 upland cotton wild type seeds are sterilized with 75% alcohol for 2 min, 10% hydrogen peroxide for 12 min, and then inoculated in germination medium (MSB + 15 g / L glucose + 2.3 g / L Gelrite, pH 6.1) after 5 times of sterile water rinsing, and cultured at 30°C in the dark until the hypocotyls are 10-12 cm long.

[0101] Agrobacterium infection: the activated Agrobacterium liquid is resuspended in resuspension medium (containing 100 μmol / L acetyl-syringone) after centrifugation until the OD 600=0.8; the hypocotyls were cut into 0.8-1 cm segments, infected for 40 min, and co-cultivated in co-cultivation medium (containing 100 μmol / L acetosyringone) in the dark for 48 h.

[0102] Screening and regeneration: transfer to selection medium, subculture every 25 d, and transfer to differentiation medium after green somatic embryos are formed, and cultivate under light (16 h / 8 h) until seedlings are rooted.

[0103] All the above operations must be completed under strict sterile conditions.

[0104] Regenerated plant planting: transplant the healthy regenerated cotton seedlings into planting pots, and manage them in the greenhouse until the cotton fibers and seeds are mature. Harvest the T0 generation genetically edited cotton seeds, continue to plant the T1 generation, and perform GUS staining and Hi-TOM sequencing to screen and identify effective edited lines as subsequent analysis materials.

[0105] Example 4: Identification of genetically edited plants

[0106] Genotype identification:

[0107] Extraction of DNA from leaf tissue of T1 generation edited cotton plants: select 0.5-1 g of young cotton leaves, quickly grind them into fine powder in liquid nitrogen, and extract genomic DNA using the EASY spin plant DNA rapid extraction kit of Aidley Company. The extraction method is strictly in accordance with the steps on the instruction manual. The quality of the DNA is detected by non-denaturing agarose gel electrophoresis.

[0108] The primer is used for amplification, and the primer sequence is as follows: the primer is synthesized by Huada Gene Company:

[0109] GhWRKY46-Check-F (SEQ ID NO: 13): 5'-ggagtgagtacggtgtgcAGCAGCAAGTTGAAGAGAAAC-3';

[0110] GhWRKY46-Check-R (SEQ ID NO: 14): 5'-gagttggatgctggatggCTGAAGCTGGTGCAGAGATTA-3';

[0111] The amplification program is as follows: 95 °C pre-denaturation for 3 min; 95 °C denaturation for 20 s, 56 °C annealing for 20 s, 72 °C extension for 30 s, a total of 40 cycles.

[0112] Amplification system (30 μL): 2x Taq Master Mix (Dye Plus) (Novozyme P112-03), 5 μmol / L of each primer 1 μL, template DNA about 100 ng, and ddH2O to 30 μL.

[0113] Post-amplification detection: the amplification product and 5 μL DNA Marker (1 kb Ladder) were loaded on a 1% gel electrophoresis, 1xTAE buffer, 120V constant voltage, 22min, and the band position of about 300bp was observed as the correct product under the gel imaging system, which met the requirements of Hi-TOM sequencing.

[0114] The target region was amplified, and the amplification product was verified by Hi-TOM (High-Throughput Identification of Targeted Mutations) sequencing to determine the mutation type (HI-TOM sequencing was completed by China Rice Research Institute, order submission website http: / / 121.40.237.174 / Hi-TOM / ).

[0115] Sequencing result analysis:

[0116] The original sequencing data was subjected to quality control, and the read length with low quality, linker contamination and the like was removed to improve the data quality. The pre-processed sequencing read length was aligned with the wild type cotton GhWRKY46 gene reference sequence to determine the editing site and editing type (such as insertion, deletion, base substitution and the like), the proportion of the number of edited sequences to the total sequencing sequence number was counted, the editing efficiency of GhWRKY46 gene in different plants or samples was calculated, in addition, the possible off-target sites were predicted by bioinformatics method, the sequencing data was aligned and analyzed with the predicted off-target sites, and whether there was off-target effect in the gene editing process was evaluated. Through the above process, the editing condition of cotton GhWRKY46 gene can be comprehensively and accurately detected by using Hi-TOM technology, and the homozygous mutation rate of GhWRKY46 gene in T1 generation gene editing plants can reach 70% to 80%, and no off-target effect is detected (such as Figure 3 A). The present application selects GhWRKY466 gene (Gh_D07G1384) homozygous mutant line as subsequent research.

[0117] Screening of homozygous mutant line without exogenous gene insertion

[0118] The gene editing vector provided by the present application fuses CaMV35S promoter-reporter gene GUS, which can be used for GUS staining method to analyze whether the exogenous gene is inserted into the transgenic line, and the GUS staining method needs to use GUS base fluid and X-Gluc using fluid as reagents.

[0119] The GUS base liquid formula is: 50 mM PBS (pH 7.0, containing 0.78 g / 100 ml of NaH2PO4, 1.791 g / 100 ml of Na2HPO4) dissolving 10 mM of EDTA-2Na (372 mg), 0.1% of Triton-100 (100 μL), 0.5 mM of potassium ferricyanide (16.5 mg), 0.5 mM of potassium ferrocyanide (21.1 mg), and then constant volume to 100 ml.

[0120] The X-Gluc using liquid: mix 50 μL X-Gluc stock solution with 950 μL GUS base liquid, and store at -20℃ for standby. Take the leaf of the GhWRKY46 homozygous mutant strain of appropriate size and place it in the staining solution, incubate at 37℃ overnight or incubate for 1-24 h until the desired staining effect appears. The Cas9 protein in the CRISPR / Cas9 system is introduced by an exogenous expression vector. Under normal circumstances, the homozygous mutant strain without exogenous insertion should not contain the Cas9 protein coding sequence. The genomic DNA of the GhWRKY46 homozygous editing strain is amplified by PCR using specific primers designed for the Cas9 gene. The primer sequences are shown in SEQ ID NO: 15 and SEQ ID NO: 16.

[0121] Cas9-Chenck primer F (SEQ ID NO: 15): 5'-cacagcatcaagaagaacctg-3';

[0122] Cas9-Chenck primer R (SEQ ID NO: 16): 5'-cctcgaacagctggttgtag-3';

[0123] If a band can be amplified, it indicates that the plant still contains the exogenous Cas9 gene; if there is no band, it may be a homozygous mutant strain without exogenous insertion. The amplification results in this embodiment are shown in FIGS. 1A and 1B. Figure 3 As shown in FIGS. 1A and 1B, the 5th and 6th strains in ko4 and the 7th and 8th strains in ko5 are homozygous mutant strains without exogenous insertion.

[0124] Example 5 Analysis of mature fiber secondary wall thickness of wild type and gene edited plants

[0125] The mature fibers of the genetically edited cotton described in Embodiment 4 at the boll opening stage (about 40-60 days after flowering) were collected as the experimental group. At the same time, the mature fibers of wild type YZ-1 Gossypium hirsutum at the same period were collected as the control group. At least 3 cotton plants were selected in each group, and the fibers of 3-5 bolls of each plant were mixed as one sample. After the fiber samples were fixed overnight with FAA fixing solution (5 mL 38% formaldehyde, 5 mL glacial acetic acid and 90 mL 70% alcohol), they were dehydrated with 95% and 100% alcohol in turn, and then dehydrated twice with anhydrous ethanol:tert-butanol (volume ratio 1:1) and infiltrated with tert-butanol three times. Then, the fibers were infiltrated with tert-butanol:paraffin (volume ratio 1:1) and pure paraffin three times in a 65°C incubator. After adjusting the position of the fibers in the embedding box, the surface paraffin was solidified by pulling the two sides of the embedding box and blowing air, and then quickly immersed in ice water until the paraffin was completely solidified. The wax block was taken out, flattened and fixed on a microtome, and then cross-sectioned at a thickness of 10 μm. The wax tape was placed on a glass slide coated with polylysine adhesive, and then dried at 37°C. Then, the fibers were deparaffinized with xylene substitute, rehydrated with gradient alcohol, stained with 0.1% fast green for 30 seconds, and then quickly washed with 95% alcohol. Then, the fibers were dehydrated with gradient alcohol and xylene substitute, and then dropped with Canada balsam. Finally, the fibers were photographed under a microscope, and the fiber cell wall thickness was measured using ImageJ 2.0 software. The differences in secondary wall thickness between the genetically edited strains and the wild type were compared using students’st test. If p<0.05, the difference was significant; if p<0.01, the difference was extremely significant.

[0126] From the morphology, the fiber cells of GhWRKY46 transgenic strains ko4 and ko5 were mostly sunken and irregular in shape compared to the wild type Figure 4 A). Further measurement of the thickness of the fiber cell wall showed Figure 4 B) The cell wall thickness of the genetically edited mature fibers was significantly lower than that of the wild type. The fiber cell wall thickness of strain ko4 was 2.785±0.96 μm, and that of strain ko5 was 2.934±1.04 μm, which was 46.8% and 43.9% lower than that of the wild type cotton fiber cell wall (5.23±0.80 μm), respectively.

[0127] The above data show that knocking out the GhWRKY46 gene can inhibit the deposition of secondary wall of cotton fiber, and cause abnormal morphogenesis of fiber cells. The secondary wall thickness and softness of cotton fiber are important indicators for measuring the quality of cotton fiber, and they are related to each other and jointly affect the textile applicability of fiber. If the secondary wall is too thick (such as more than 4 μm), the fiber will be rigid and the softness will decrease; if it is too thin (such as less than 2.5 μm), the strength will be insufficient. The statistical results show that the secondary wall thickness of the GhWRKY46 knockout strain is thinner, and it has softness and strength. The method of the present application is simple and easy to operate, the effect is significant, and it has good market prospect.

[0128] Example 6 Mature fiber length analysis of wild type and gene edited plants

[0129] The experimental group and the control group of this example are the same as the experimental group and the control group in Example 5. The upper and middle cotton bolls of the experimental group and the control group are taken, 10 cotton plants are selected from each group, 10 cotton bolls are taken from each cotton plant, and 5 seeds are randomly taken from each cotton boll. After the fibers are combed, the fiber length is measured (such as Figure 5 A), and the mature fiber length of the gene edited cotton and the wild type cotton of the control group is counted (such as Figure 5 B). The results show that the mature cotton fiber length of the gene edited strains ko4 and ko5 is 3.25±0.075 cm and 3.205±0.08 cm, respectively, and the mature fiber length of the wild type is 2.76±0.040 cm. Compared with the wild type, the fiber length of ko4 and ko5 is significantly increased by 17.48% and 15.90%, respectively.

[0130] Example 7 Mature fiber quality analysis of wild type and gene edited plants

[0131] The experimental group and the control group of this example are the same as the experimental group and the control group in Example 5. At the normal boll opening period of cotton (40-60 days after flowering), cotton bolls are picked on sunny days to avoid the influence of rain and fog on fiber quality. 5-10 cotton plants are randomly selected, and 5-10 middle cotton bolls are taken from each plant. The cotton bolls taken from the experimental group and the control group are mixed to form representative samples, and impurities (such as cotton leaves and boll shells) are removed. The fibers are separated from the cotton seeds to form lint and seed cotton samples. The cotton fiber samples are sent to the Cotton Quality Supervision and Inspection Testing Center of the Ministry of Agriculture and Rural Affairs (Anyang) for testing. According to ASTM D586795 "HVI900 Large Capacity Fiber Tester Test Method", HFT9000 is used to test the upper half average length, uniformity index, breaking specific strength, elongation, and micronaire value under the environmental conditions of temperature 20℃ and relative humidity 65%. The results are shown in Table 2.

[0132] Table 2 Comparison of fiber quality traits of GhWRKY46 gene edited cotton and control

[0133]

[0134]

[0135] Note: In the table, ** indicates a significant difference compared with the WT group (P < 0.01).

[0136] According to Table 2, it can be seen that the maturity fiber uniformity indexes of the ko4 and ko5 strains in 2023 have no obvious difference with the wild type control. The upper half average length of the mature fiber of the ko4 and ko5 strains is increased by 9.5% and 7% respectively compared with the wild type, the breaking specific strength is increased by 8.6% and 1.9% respectively compared with the wild type, the micronaire value is reduced by 12% and 6% respectively compared with the wild type, and the elongation is increased by 3.0% compared with the wild type. The fiber uniformity indexes of the ko4 and ko5 gene edited strains in 2024 have no obvious difference with the wild type control. The upper half average length of the fiber of the ko4 and ko5 gene edited strains is increased by 19.7% and 15.5% respectively compared with the wild type, the breaking specific strength is increased by 18% and 9% respectively compared with the wild type, the micronaire value is reduced by 10% and 24% respectively compared with the wild type, and the elongation is increased by 4.8% and 3.2% respectively compared with the wild type. From the fiber quality detection results of two consecutive years, the GhWRKY46 gene edited strains are good in fiber length, elongation, breaking specific strength and micronaire value quality indexes. Compared with the wild type, the differences of each quality trait index in 2023 and 2024 are small, and the excellent quality index inheritance is stable, so it can be used as an excellent cotton fiber strain for promotion.

[0137] In summary, the present application obtains a GhWRKY46 gene for regulating cotton fiber frequency through screening. Through precise editing of the endogenous GhWRKY46 gene of Gossypium hirsutum, the gene edited cotton has increased fiber length, thinned secondary wall thickness, reduced micronaire value and improved breaking specific strength, and the cotton fiber quality is significantly improved, and an excellent germplasm resource of cotton with improved fiber length and strength is obtained. The gene provided by the present application makes the comprehensive quality of cotton fiber significantly better than the wild type, and has important agricultural application value.

[0138] Although the above embodiment has made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained as the present embodiment without creativity, which all belong to the protection scope of the present application.

Claims

1. Application of GhWRKY46 gene in regulating cotton fiber quality, characterized in that, An amino acid sequence coded by the GhWRKY46 gene is shown as SEQ ID NO:

1.

2. Use according to claim 1, wherein A nucleotide sequence of the GhWRKY46 gene is shown as SEQ ID NO:

2.

3. The use according to claim 1, wherein The cotton fiber quality regulation comprises knocking out or reducing the expression of the GhWRKY46 gene in cotton, and improving the cotton fiber quality.

4. Use according to any one of claims 1 to 3, characterized in that, The cotton fiber quality comprises one or more of the following: fiber length, fiber elongation, breaking specific strength, and micronaire value.

5. Application of a biological material for negatively regulating the GhWRKY46 gene in regulating the cotton fiber quality.

6. A biological material for down-regulating the expression of a cotton GhWRKY46 gene, characterized in that, The biological material comprises one or more of the following: a transformant of the GhWRKY46 gene, an sgRNA, a primer pair of the sgRNA, a recombinant vector containing the sgRNA, and a recombinant microorganism containing the sgRNA. The transformant of the GhWRKY46 gene comprises a transformant ko4 and a transformant ko5.

7. The biomaterial of claim 6, wherein The transformant ko4 is: deletion of CCA at positions 1075-1077 and deletion of ATGGGCAAC at positions 1079-1087 of the GhWRKY46 gene. The transformant ko5 is: insertion of a G base at position 1078 of the GhWRKY46 gene. A sequence of the sgRNA is shown as SEQ ID NO:

7.

8. The biomaterial of claim 6, wherein A primer pair of the sgRNA is shown as SEQ ID NO:9 and SEQ ID NO:

10.

9. The biomaterial of claim 6, wherein The cotton fiber quality regulation comprises knocking out or reducing the expression of the GhWRKY46 gene in cotton, and improving the cotton fiber quality.

10. A breeding method for improving the quality of cotton fibers, characterized by, The cotton fiber quality comprises one or more of the following: fiber length, fiber elongation, breaking specific strength, and micronaire value.

5. Application of a biological material for negatively regulating the GhWRKY46 gene in regulating the cotton fiber quality. The biological material comprises one or more of the following: a transformant of the GhWRKY46 gene, an sgRNA, a primer pair of the sgRNA, a recombinant vector containing the sgRNA, and a recombinant microorganism containing the sgRNA. The transformant of the GhWRKY46 gene comprises a transformant ko4 and a transformant ko5. The transformant ko4 is: deletion of CCA at positions 1075-1077 and deletion of ATGGGCAAC at positions 1079-1087 of the GhWRKY46 gene. The transformant ko5 is: insertion of a G base at position 1078 of the GhWRKY46 gene. A sequence of the sgRNA is shown as SEQ ID NO:

7. A primer pair of the sgRNA is shown as SEQ ID NO:9 and SEQ ID NO:

10. The cotton fiber quality regulation comprises knocking out or reducing the expression of the GhWRKY46 gene in cotton, and improving the cotton fiber quality. The cotton fiber quality comprises one or more of the following: fiber length, fiber elongation, breaking specific strength, and micronaire value.

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