Cotton hd-zip transcription factor ghHB6 gene and application

Abstract

The application belongs to the technical field of cotton molecular breeding, and particularly relates to a cotton HD-Zip transcription factor GhHB6 gene and application. The application provides a biological material for knocking out GhHB6 a gene and / or knocking out GhHB6 a gene and application of the biological material in improving cotton fiber quality, wherein the GhHB6 gene comprises an amino acid sequence as shown in SEQ ID NO: 1. It is verified that after knocking out the GhHB6 gene in cotton, indexes such as fiber length, breaking specific strength, elongation rate and regularity are significantly improved. Therefore, the GhHB6 GhHB6 gene is an excellent target for genetic improvement of cotton fiber quality, and editing the gene by using CRISPR technology can effectively and comprehensively improve the comprehensive quality of cotton fiber.

Description

Technical Field

[0001] This invention belongs to the field of cotton molecular breeding technology, specifically relating to a cotton HD-Zip transcription factor. GhHB6 Genes and their applications. Background Technology

[0002] cotton( Gossypium spp Cotton is the world's most important natural fiber crop, hailed as "white gold." With rising consumption levels and increasing market demand for high-end textiles, cultivating new cotton varieties with longer, stronger, and finer fibers has become one of the core goals of agricultural biotechnology. Cotton fibers are formed from the epidermal cells of the ovule through single-cell differentiation, extreme elongation, and secondary wall thickening. This complex developmental process is precisely regulated by a multi-level gene network, with transcription factors playing a crucial role as the core regulatory hub.

[0003] The HD-ZIP (homeodomain-leucine zipper) family plays a crucial role in regulating cotton growth and development; however, significant gaps remain in our understanding of the HD-ZIP family, particularly its downstream precise regulatory networks and functional diversity. Most research focuses on a few key genes (such as...). GhHOX3 However, little is known about the functional redundancy, synergistic interaction mechanisms, and potential regulatory role in the synthesis of secondary fiber walls of other family members, which greatly limits the ability of this family of genes to be used for molecular breeding in this field.

[0004] Therefore, the function of the HD-ZIP transcription factor family genes in regulating cotton fiber quality needs to be further explored. Summary of the Invention

[0005] To address the lack of genes in existing technologies that regulate cotton fiber growth and improve cotton fiber quality, this invention provides a cotton HD-Zip transcription factor. GhHB6 Genes and their applications, specifically including the following technical solutions:

[0006] This invention provides knockout GhHB6 Gene and / or knockout GhHB6 The application of gene-based biomaterials in improving cotton fiber quality, the aforementioned GhHB6 The gene includes an amino acid sequence as shown in SEQ ID NO:1.

[0007] Preferably, the GhHB6 The nucleotide sequence of the gene is shown in SEQ ID NO:2.

[0008] Preferably, the improved cotton fiber quality includes any one or more of the following:

[0009] 1) Increase the length of cotton fibers;

[0010] 2) Improve the breaking strength of cotton fibers;

[0011] 3) Improve the uniformity of cotton fibers.

[0012] Preferably, the knockout GhHB6 Biological materials for genes include sgRNA, knockout GhHB6 Recombinant expression vectors and knockout of genes GhHB6 Microorganisms that regenerate genes.

[0013] Preferably, the sgRNA comprises the sequence shown in SEQ ID NO: 3.

[0014] Preferably, the knockout GhHB6 The recombinant expression vector of the gene includes a backbone vector; the backbone vector includes the pRGEB32-GhU6.7 vector.

[0015] Preferably, the knockout GhHB6 The recombinant microorganisms of the gene include basic microorganisms; said basic microorganisms include Agrobacterium EHA105.

[0016] The present invention also provides a method for improving the quality of cotton fibers, comprising the following steps:

[0017] Knock out the target plant GhHB6 Genes that result in plants with improved cotton fiber quality;

[0018] The GhHB6 The gene includes an amino acid sequence as shown in SEQ ID NO:1.

[0019] Preferably, the cotton includes upland cotton.

[0020] Preferably, the improved cotton fiber quality includes any one or more of the following:

[0021] 1) Cotton fiber length elongation;

[0022] 2) The breaking strength of cotton fibers is improved;

[0023] 3) Improved uniformity of cotton fibers.

[0024] The beneficial effects of this invention are as follows:

[0025] This invention uses CRISPR-Cas gene editing technology to knock out or inhibit [the growth of certain substances] in cotton. GhHB6This invention utilizes genetics to cultivate new cotton varieties with superior fiber quality, characterized by longer, more uniform, and stronger fibers. It provides an effective way to enhance the economic value of cotton and improve the quality of cotton fiber textiles. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0027] Figure 1 A map of the pRGEB32-GhU6.7-GhHB6 mutant plasmid vector provided by this invention;

[0028] The vector backbone pRGEB32-GhU6.7 contains two independent resistance gene systems: one for screening colonies containing the plasmid in bacteria, and the other for screening successfully transformed struts in plants. Both systems use the same antibiotic—kanamycin.

[0029] Figure 2 A gel image showing the Cas9-positive detection results of the mutant transgenic material provided by this invention;

[0030] Figure 3 A gel image showing the NPTII positive test results of the mutant transgenic material provided by this invention;

[0031] Figure 4 A gel image showing the sgRNA positive detection results of the mutant transgenic material provided by this invention;

[0032] in, Figures 2-4 In the middle, the leftmost lane shows the results of marker electrophoresis (from top to bottom: 100, 250, 500, 750, 1000, 2000, 3000, 5000 bp). CR-1 represents the GhHB6 knockout line, N represents the wild-type recipient material, and P represents the positive plasmid as a positive control.

[0033] Figure 5 The Hi-TOM test results chart is used to analyze the editing efficiency test results;

[0034] Figure 6 The result of hand-combing the transgenic material fiber provided by this invention;

[0035] Figure 7 A bar chart showing fiber length at different stages of the mutant;

[0036] Where * represents P <0.05, ** indicates P <0.01, *** indicates P <0.001. Detailed Implementation

[0037] This invention provides a knockout GhHB6 Gene and / or knockout GhHB6 The application of gene-based biomaterials in improving cotton fiber quality, the aforementioned GhHB6 The gene includes an amino acid sequence as shown in SEQ ID NO:1.

[0038] SEQ ID NO:1:MKRLGSSDSLSALMSICPTSDERSPRNNNVYSREFQSmLEGFDEEGCFEELTVGHGGGAKKRRLSVDQVKALEKNFEVENKLEPDRKSKLAQELGLQPRQVAVWFQNRRARWKTKQLERDYGVLKTSYESLKLNYSNLRHE NEDLLKQISELKAKLIEPPPGSSLGTSLEPAEVNCESFNDGGNGNGSVGGGGDTVFPDLKDGSSDSDSSAILNEENNSGGDMEGGSPNNGAASSPSSMNNCFQLFKTTYQTPQFVKMEEHDFISSTADEVCNFFSDEQAPSLQWWI.

[0039] As one implementation method, the present invention described GhHB6 The nucleotide sequence of the gene is shown in SEQ ID NO:2.

[0040] SEQ ID NO:2:

[0041] 5'--3'.

[0042] As one embodiment, the improved cotton fiber quality of the present invention includes any one or more of the following: 1) increasing cotton fiber length; 2) improving cotton fiber breaking strength; 3) improving cotton fiber uniformity.

[0043] As one implementation method, the knockout GhHB6 Biological materials for genes include sgRNA, knockout GhHB6 Recombinant expression vectors and knockout of genes GhHB6 Recombinant microorganisms of genes. As one embodiment, the sgRNA comprises the sequence shown in SEQ ID NO:3. As one embodiment, the knockout... GhHB6 The recombinant gene expression vector includes a backbone vector. In one embodiment, the backbone vector includes a plasmid vector. In one embodiment, the plasmid vector includes the pRGEB32-GhU6.7 vector. In one embodiment, the restriction enzyme site on the pRGEB32-GhU6.7 vector is BsaI enzyme. This invention references the cotton genome sequence and the CRISPR-P website http: / / crispr.hzau.edu.cn / cgi-bin / CRISPR2 / CRISPR to search for targets, designs primers for PCR amplification, and uses BsaI enzyme to target pRGEB32-GhU6.7 (Wang et al. High efficient multisites genome editing in allotetraploidcotton (Gossypium hirsutum) using CRISPR / Cas9 system. Plant Biotechnol J.2018 Jan;16(1):137-150. doi: 10.1111 / pbi.12755. Epub 2017 Jun 20. PMID:28499063; PMCID: The empty vector (PMC5785356.) was digested with enzymes, followed by gel electrophoresis and purification. The target fragment was then ligated to the linearized expression vector using Exnase. The constructed vector was named pRGEB32-GhU6.7-GhHB6. The vector map is shown below. Figure 1 As shown.

[0044] As one implementation method, the knockout GhHB6The recombinant microorganisms for the gene include basic microorganisms; said basic microorganisms include Agrobacterium EHA105. This invention preferably involves heat shock transformation of Escherichia coli TOP10, identification of positive clones by PCR, propagation and plasmid extraction, and transformation of the plasmids into competent Agrobacterium strain EHA105 cells by electroporation. Clones identified positive by PCR are preserved for later use. Agrobacterium EHA105 containing pRGEB32-GhU6.7-GhHB6 is propagated, and then the T-DNA fragment is transformed into cotton hypocotyls using Agrobacterium-mediated genetic transformation. Regenerated plants are obtained through tissue culture. Primers designed targeting the Cas9 protein are used to amplify the gene-edited plants by PCR to identify positive plants; primers designed targeting the NPTII resistance gene are used to amplify the gene-edited plants by PCR to identify positive plants; primers designed targeting the sequences at both ends of the sgRNA on the pRGEB32-GhU6.7-GhHB6 vector are used to amplify the gene-edited plants by PCR to identify positive plants. PCR amplification of the transgenic positive materials produces the corresponding bands. Hi-tom high-throughput sequencing technology was used to analyze the editing sites of mutants.

[0045] This invention clones from cotton to GhHB6 Cloned using this invention GhHB6 Genes were constructed into CRISPR vectors, and functional verification was performed using Agrobacterium-mediated genetic transformation. Knockout was confirmed. GhHB6 Genes and fiber quality have been comprehensively improved: fibers have been significantly elongated, breaking strength has been substantially enhanced, and elongation and uniformity have also been effectively improved and optimized.

[0046] The present invention also provides a method for improving the quality of cotton fibers, comprising the following steps:

[0047] Knock out the target plant GhHB6 Genes that result in plants with improved cotton fiber quality;

[0048] The GhHB6 The gene includes an amino acid sequence as shown in SEQ ID NO:1.

[0049] In one embodiment, the cotton includes upland cotton. In another embodiment, the upland cotton includes the upland cotton strain JIN668.

[0050] As one implementation method, the knockout target plant contains GhHB6 Gene expression can be achieved by converting cotton with sgRNA, recombinant expression vectors, or recombinant microorganisms as described above.

[0051] As one implementation, the improved cotton fiber quality includes any one or more of the following:

[0052] 1) Cotton fiber length elongation; 2) Cotton fiber breaking strength increased; 3) Cotton fiber uniformity improved.

[0053] The composition of the culture medium used in this invention is as follows:

[0054] MS medium: macroelements (KNO3 1.9 g / L, KH2PO4 0.17 g / L, MgSO4·7H2O 0.37 g / L, CaCl2·2H2O 0.44 g / L), microelements (KI 0.83 mg / L, H3BO3 6.2 mg / L, MnSO4·4H2O 22.3 mg / L, Z ... 4· 7H2O 8.6 mg / L, Na2MoO4·2H2O 0.25 mg / L, CuSO4·5H2O 0.025 mg / L, CoCl2 0.025 mg / L, iron salts (Na2·EDTA 37.3 mg / L, FeSO4·7H2O 27.8 mg / L), organic components (inositol 100 mg / L, glycine (Gly) 2 mg / L, VB1 0.1 mg / L, VB6 0.5 mg / L, VB5 0.5 mg / L).

[0055] MGL medium: tryptone 5 g / L, NaCl 5 g / L, MgSO4·7H2O 0.1 g / L, KH2PO4 0.25 g / L, mannitol 5 g / L, glycine 1 g / L, and distilled water to make up to 1 L.

[0056] 2,4-D induction medium: MS medium was used as the basal medium, with the addition of 0.1 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), 0.1 mg / L cytokinin (KT), 30 g / L glucose, and 2.5 g / L Phytagel. The volume was then adjusted to 1 L with distilled water. The pH was adjusted to 5.9.

[0057] Embryo differentiation medium: MS medium was used as the basal medium, supplemented with 1.9 g / L KNO3, 0.1 mg / L KT, 30 g / L glucose, 1.0 g / L glutamine (Gln), 0.5 g / L asparagine (Asn), and 2.5 g / L phytogel, and diluted to 1 L with distilled water. The pH was adjusted to 5.9.

[0058] 1 / 2MS medium: KNO3 0.95 g / L, KH2PO4 0.085 g / L, MgSO4·7H2O 0.185 g / L, CaCl2·2H2O 0.22 g / L.

[0059] Rooting medium: Use 1 / 2 MS medium as the basal medium, add 15 g / L glucose and 2.5 g / L Phytagel, and make up to 1 L with distilled water. Adjust the pH to 5.9.

[0060] To further illustrate the present invention, the following description, in conjunction with the accompanying drawings and embodiments, describes a cotton HD-Zip transcription factor provided by the present invention. GhHB6 The genes and their applications are described in detail, but they should not be construed as limiting the scope of protection of this invention.

[0061] Example 1: Construction of the GhHB6 CRISPR vector

[0062] Referring to the CRISPR-P website http: / / crispr.hzau.edu.cn / cgi-bin / CRISPR2 / CRISPR, target searches were conducted using gene sequences to select sgRNA sequences located in exon regions with high scores, as shown in SEQ ID NO:3:

[0063] SEQ ID NO: 3: 5'-GGACTTGAAAGACGGATCAT-3';

[0064] The primers used for constructing the design vector are shown below:

[0065] pRGEB32-GhU6.7-F (SEQ ID NO:4):

[0066] 5'-AAGCATCAGATGGGCAAACAAAGCACCAGTGGTCTAG-3';

[0067] CR-HB6-R (SEQ ID NO:5):

[0068] 5'-TTCTAGCTCTAAAACGGACTTGAAAGACGGATCATTGCACCAGCCGGGAAT-3'.

[0069] In this embodiment, a single sgRNA sequence is recombined into the pRGEB32-GhU6.7 vector. Specifically, the preparation method is as follows:

[0070] PCR amplification of sgRNA was performed using primers pRGEB32-GhU6.7-F and CR-HB6-R to obtain PCR products containing trRNA-gRNA. Then, the empty pRGEB32-GhU6.7 vector was digested with BsaI restriction endonuclease at 37℃ for 6 h. After digestion, gel electrophoresis was performed to recover the large fragment of the pRGEB32-GhU6.7 empty vector. The target fragment from the second PCR amplification was then infused with the linearized expression vector using Exnase enzyme. The constructed vector was named pRGEB32-GhU6.7-GhHB6, and its vector pattern is shown below. Figure 1 As shown, the reaction product was used to transform E. coli competent cells TOP10. After 10-12 h, single clones were picked for PCR positive detection.

[0071] The primers for a positive test are as follows:

[0072] U6-7S (SEQ ID NO:6): 5'-TGTGCCACTCCAAAGACATCAG-3';

[0073] CR-HB6-R (SEQ ID NO:5): 5'-TTCTAGCTCTAAAACGGACTTGAAAGACGGATCATTGCACCAGCCGGGAAT-3'.

[0074] The PCR reaction system consisted of 20 μL: 16 μL ddH2O; 2 μL 10X EasyTaq Buffer; 0.4 μL dNTP; 0.2 μL Forward / Reverse Primer; 0.2 μL EasyTaq enzyme; and 1 μL bacterial culture.

[0075] The PCR reaction conditions were as follows: 95℃ pre-denaturation for 5 min; 95℃ for 30 sec, 58℃ for 30 sec, 72℃ for 20 sec, 28 cycles; 72℃ extension for 5 min.

[0076] Positive clones were amplified and plasmids were extracted to obtain the mutant plasmid pRGEB32-GhU6.7-GhHB6 for transformation. Next, 100 ng of the plasmid was electroporated into strain EHA105. After incubation at 28°C for 36 h, single clones were picked for PCR detection.

[0077] The primers for positive detection are shown in SEQ ID NO:6 and SEQ ID NO:5.

[0078] The PCR reaction system consisted of 20 μl: 16 μL ddH2O; 2 μL 10X EasyTaq Buffer; 0.4 μL dNTP; 0.2 μL Forward / Reverse Primer; 0.2 μL EasyTaq enzyme; and 1 μL bacterial culture.

[0079] The PCR reaction conditions were as follows: 95℃ pre-denaturation for 5 min; 95℃ for 30 sec, 58℃ for 30 sec, 72℃ for 20 sec, 28 cycles; 72℃ extension for 5 min.

[0080] Example 2 GhHB6 Genetic transformation of knockout vectors and screening and identification of knockout lines

[0081] A. Agrobacterium-mediated genetic transformation

[0082] The test material was upland cotton variety (JIN668). Plump and uniformly sized JIN668 seeds were selected, the seed coat was removed, and the seeds were sterilized with 0.1% mercuric chloride solution for 10 min, shaking continuously during sterilization. After sterilization, the seeds were rinsed 3 times with sterile water, placed on the surface of MS medium, and incubated in the dark at 30℃ for 1 day. The seedlings were then supported and incubated in the dark for another 5 days. At this time, the cotton cotyledons reached the top of the bottle, and sterile seedlings were obtained.

[0083] The glycerol tube containing the target gene (i.e., the pRGEB32-GhU6.7-GhHB6 vector constructed in Example 1) of strain EHA105, which was stored in a -80℃ freezer, was thawed on ice. 10 μl of strain EHA105 containing the target gene was added to 2 mL of LB liquid containing 100 mg / L kanamycin and cultured at 28℃ with shaking for 1 day to activate the strain.

[0084] The activated bacterial culture was inoculated into 20 mL of fresh LB medium containing 100 mg / L kanamycin, with an inoculation volume of 20 μL. The culture was incubated overnight at 28°C with shaking. One mL of the turbid bacterial culture was transferred to a 2 mL sterile centrifuge tube and centrifuged at 12000 rpm for 30 s to collect the bacterial cells. The cells were then resuspended in 20 mL of MGL medium containing 50 mg / L acetylsyringone (AS) and incubated at 28°C with shaking for 30–40 min for use in infecting hypocotyls.

[0085] The specific steps of Agrobacterium-mediated transformation of cotton hypocotyls are as follows:

[0086] (1) In a clean bench, take 30 sterile seedlings, cut the hypocotyl into 0.5-0.8 cm segments on sterile filter paper and inoculate into 50 mL sterile conical flasks. Add the activated Agrobacterium tumefaciens EHA105 bacterial solution containing the target vector pRGEB32-GhU6.7-GhHB6 and inoculate for 5 min, shaking several times during the process.

[0087] (2) Pour off the bacterial solution, place the hypocotyl on sterile filter paper to absorb the surface bacterial solution, place it on a clean bench and blow it for 10-15 minutes, then inoculate it onto 2,4-D induction medium without antibiotics, and co-culture at 19°C in the dark for 36 h.

[0088] (3) After co-culture, hypocotyl segments were inoculated into 2,4-D induction medium containing 100 mg / L kanamycin and 100 mg / L cephalosporin, and cultured at 28°C under weak light (cold light source 135 μmol m) -2 s -1 Cultured under the same conditions, and continuously subcultured until embryogenic callus tissue appears;

[0089] (4) Embryogenic callus tissues were successively inoculated into embryo differentiation medium and cultured until somatic embryos matured. Mature cotyledon embryos were inoculated into rooting medium to germinate until complete plants were obtained, thus obtaining transgenic plants.

[0090] B. Identification of transgenic plants

[0091] (1) Positive detection of transgenic T0 generation plants

[0092] Genomic DNA was extracted from the young leaves of transgenic plants using a plant genomic DNA extraction kit from Tiangen Biotech (Beijing) Technology Co., Ltd. (see the kit's instruction manual for specific operating procedures). The DNA was then subjected to PCR detection using the three primer pairs described below to verify the presence of corresponding T-DNA insertion. Positive detection results for transgenic plants were as follows: Figures 2-4 As shown, the corresponding T-DNA was successfully introduced into the transgenic plant constructed in this invention.

[0093] The PCR amplification primers for Cas9-positive detection of transgenic materials are shown below:

[0094] Cas9-F (SEQ ID NO:7): 5'-GCTTGTGCGTTTCGATTTGA-3';

[0095] Cas9-R (SEQ ID NO:8): 5'-CCGCTCGTGCTTCTTATCCT-3'.

[0096] The PCR amplification primers for NPTII positive detection of transgenic materials are shown below:

[0097] NPTII-F (SEQ ID NO:9): 5'-GCTTGGGTGGAGAGGCTATTC-3';

[0098] NPTII-R (SEQ ID NO: 10): 5'-GAAGAACTCGTCAAGAAGGCG-3'.

[0099] The PCR amplification primers for sgRNA positivity detection of transgenic materials are shown below:

[0100] sgRNA-F (SEQ ID NO:11): 5'-TATAAGCGAAAGAAGCATCAGA-3';

[0101] sgRNA-R (SEQ ID NO: 12): 5'-GACCCGAATTTGTGGACC-3'.

[0102] The PCR reaction system consisted of 20 μl: 16 μL ddH2O; 2 μL 10X EasyTaq Buffer; 0.4 μL dNTP; 0.2 μL Forward / Reverse Primer; 0.2 μL EasyTaq enzyme; and 1 μL DNA.

[0103] PCR reaction conditions: 95℃ pre-denaturation for 5 min; 95℃ for 30 sec, 58℃ for 30 sec, 72℃ for 50 sec, 28 cycles; 72℃ extension for 5 min.

[0104] Example 3 GhHB6 CRISPR knockout strain editing efficiency detection

[0105] The method for extracting DNA from cotton leaves is the same as in Example 2. Because CRISPR-Cas9 technology can result in multiple editing types on a single plant, and Sanger sequencing cannot identify multiple mutation types, high-throughput Hi-TOM sequencing technology was used to detect the editing efficiency of the knockout lines. The specific steps are as follows:

[0106] (1) Design primers according to the conventional PCR primer design principles (16-20 nt); the target site should be within 10-100 bp of the left or right primer. For independent single-plant materials with gene knockout, design site-specific primers with common adapter sequences (SEQ ID NO:13: 5′-ggagtgagtacggtgtgc-3′ and SEQ ID NO:14: 5′-gagttggatgctggatgg-3′) to amplify the genome sequence of the target gene in the first round of PCR.

[0107] The first round of PCR reaction system (20 μL) contains: 50 ng genomic DNA, 0.2 µL of 10 µmol / L forward and reverse primers, and 10 µL of 2×Taq Master Mix, with ddH2O added to bring the total to 20 μL.

[0108] The first round of PCR reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ for 30 sec, 58℃ for 30 sec, 72℃ for 20 sec, 29 cycles; 72℃ extension for 5 min.

[0109] (2) Using the first round of PCR products as templates, primers containing barcode and index were added for the second round of PCR amplification.

[0110] The second round PCR reaction system (20 μL) includes: 1 μL of the first round PCR product, 0.4 nmol / L of forward and reverse barcode primers, 200 nmol / L of forward and reverse index primers, and 10 µL of 2×Taq Master Mix, with ddH2O added to bring the total to 20 μL.

[0111] The second round of PCR reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ for 30 sec, 58℃ for 30 sec, 72℃ for 30 sec, 30 cycles; 72℃ extension for 5 min.

[0112] (3) The obtained second-round PCR products were mixed in equal volumes from each well and purified using a purification kit (OMEGA, D2500-2). Each mixture of 96 individual samples was sequenced using a second-generation sequencing platform, yielding 1 G of data. The data were analyzed using the Hi-TOM analysis website (http: / / www.hi-tom.net / hi-tom / ). The sequenced data were compared with the reference genome sequence to evaluate the final editing efficiency.

[0113] Using a barcode-based approach, for each individual mutant of the knockout material, a barcode is attached to the 5' end of both primers targeting the target site, via a adapter. This results in a unique barcode pair for each mutant. These barcode-containing primers are then used to amplify the target site sequences of individual mutants, constructing a mixed DNA library for high-throughput sequencing. Sequencing results are sorted according to the barcode-labeled primers to obtain the sequencing results for each individual mutant. After removing repetitive and low-quality sequences, the sequencing results are compared with a reference gene sequence to complete the process of detecting mutations at the target gene site in individual mutants.

[0114] The Barcode primers used in the detection are as follows:

[0115] F-1 (SEQ ID NO:15): 5’-GCTTGCGTTGGAGTGAGTACGGTGTGC-3’;

[0116] F-2 (SEQ ID NO:16): 5’-GCTTGTAGTGGAGTGAGTACGGTGTGC-3’;

[0117] F-3 (SEQ ID NO:17): 5’-GCTTACGCTGGAGTGAGTACGGTGTGC-3’;

[0118] F-4 (SEQ ID NO:18): 5’-GCTTCTCGTGGAGTGAGTACGGTGTGC-3’;

[0119] F-5 (SEQ ID NO:19): 5’-GCTTGCTCTGGAGTGAGTACGGTGTGC-3’;

[0120] F-6 (SEQ ID NO:20): 5’-GCTTAGTCTGGAGTGAGTACGGTGTGC-3’;

[0121] F-7 (SEQ ID NO:21): 5’-GCTTCGACTGGAGTGAGTACGGTGTGC-3’;

[0122] F-8 (SEQ ID NO:22): 5’-GCTTGATGTGGAGTGAGTACGGTGTGC-3’;

[0123] F-9 (SEQ ID NO:23): 5’-GCTTATACTGGAGTGAGTACGGTGTGC-З’;

[0124] F-10 (SEQ ID NO:24): 5’-GCTTCACATGGAGTGAGTACGGTGTGC-3’;

[0125] F-11 (SEQ ID NO:25): 5’-GCTTGTGCTGGAGTGAGTACGGTGTGC-3’;

[0126] F-12 (SEQ ID NO:26): 5’-GCTTACTATGGAGTGAGTACGGTGTGC-3’;

[0127] RA (SEQ ID NO:27): 5'-CCATCCAGCATCCAACTCAACGCACAG-3';

[0128] RB (SEQ ID NO:28): 5'-CCATCCAGCATCCAACTCACTACACAG-3';

[0129] RC (SEQ ID NO:29): 5'-CCATCCAGCATCCAACTCAGCGTACAG-3';

[0130] RD (SEQ ID NO:30): 5'-CCATCCAGCATCCAACTCACGAGACAG-3';

[0131] RE (SEQ ID NO:31): 5'-CCATCCAGCATCCAACTCAGAGCACAG-3';

[0132] RF (SEQ ID NO:32): 5'-CCATCCAGCATCCAACTCAGACTACAG-3';

[0133] RG (SEQ ID NO:33): 5'-CCATCCAGCATCCAACTCAGTCGACAG-3';

[0134] RH (SEQ ID NO:34): 5'-CCATCCAGCATCCAACTCACATCACAG-3'.

[0135] The index primers used for detection are as follows:

[0136] 2P-F-1 (SEQ ID NO:35): 5'-AATGATACGGCGACCACCGAGATCTACACAGGAACCTACACTCTTTCCCTACACGACGCTCTT-3';

[0137] 2P-R-1 (SEQ ID NO:36): 5'-CAAGCAGAAGACGGCATACGAGATCTTAGCCAGTGACTGGAGTTCAGACGTGTGCTCTT-3'.

[0138] The PCR reaction system consisted of 20 µL, with the specific components as described above.

[0139] The PCR reaction conditions were: 95℃ pre-denaturation for 5 min; 30 cycles of 95℃ for 30 sec, 58℃ for 30 sec, and 72℃ for 30 sec; extension at 72℃ for 5 min. Equal volumes of the PCR product were purified and sequenced. The results are as follows: Figure 5 As shown, this invention has successfully constructed a transgenic strain.

[0140] Example 4: Using genetically modified cotton to... GhHB6 Gene functional verification

[0141] The specific steps are as follows:

[0142] A. GhHB6 Measurement of immature fiber length in transgenic lines

[0143] In the experimental field of Huazhong Agricultural University, cotton bolls from the same location in both the transgenic and control lines (wild-type JIN668 upland cotton) were harvested at the same time point for fiber length measurement. Twenty biological replicates were performed for each line, and the collected bolls were measured at 8 DPA (days after flowering), 12 DPA, 16 DPA, and 20 DPA. Ovules from the same location on the boll were placed in boiling water and gently tapped with a glass rod to disperse the fibers. The cotton fiber length was measured using the cottonseed combing method. The specific procedures included: first, straightening the fibers along the central groove of the cottonseed; then rinsing under running water to straighten the fibers; and finally combing with a comb. Afterward, the cottonseeds were placed groove-down on a black felt board, and the length was measured with a steel ruler. The obtained data were analyzed using Prism software for multiple comparisons. The results are shown below. Figure 6 and Figure 7 It is evident that, compared to the wild type, knocking out G... hHB6 Cotton fiber elongation can be significantly promoted at 8 DPA, 12 DPA, 16 DPA and 20 DPA.

[0144] B. Determination of mature fiber length in GhHB6 knockout lines

[0145] Harvested at the same time as cotton fiber maturity. GhHB6 Fiber length was measured in bolls from the same fruiting branch location in gene knockout plants and control plants (wild-type JIN668 upland cotton), with 20 biological replicates per line. The measurement was performed using the cotton seed combing method. First, the fibers were straightened along the central ventral groove of the cotton seed, and then combed. Next, the cotton seed was placed groove-down on a black felt board, and the length was measured using a steel ruler. The obtained data were analyzed using Prism software for multiple comparisons. The results are shown below. Figure 6 It is evident that, compared to the wild type, the knockout GhHB6 It can significantly promote the elongation of cotton fibers.

[0146] C. GhHB6 Identification of mature fiber quality in knockout strains

[0147] Mature cotton bolls from the middle of the plant at the same stage were manually picked and then machine-ginned. Each fiber sample weighed approximately 10 g. The processed samples were then measured for five parameters using a High Volume Instrument (HFT9000, Premier, India). Each line was replicated at least three times, and the data were analyzed using SPSS software for multiple comparisons. The results are shown in Table 1.

[0148] Table 1 Results of Quality Identification of Mature Fibers

[0149]

[0150] Where * represents P <0.05, ** indicates P <0.01; T2 and T4 represent different generations of genetically modified cotton.

[0151] As shown in Table 1, compared with the wild type, the knockout GhHB6 The quality of the genetically modified fiber has been comprehensively improved: the fiber has been significantly elongated, the breaking strength has been substantially enhanced, and the elongation and uniformity have also been effectively improved and optimized.

[0152] In summary, this invention provides a knockout mechanism. GhHB6 Gene and / or knockout GhHB6 The application of gene-based biomaterials in cotton breeding. This invention has been verified to discover the ability to knock out genes in cotton. GhHB6 After gene modification, fiber length, breaking strength, elongation, and uniformity were all significantly improved. Therefore, the present invention... GhHB6 Genes are excellent targets for the genetic improvement of cotton fiber quality. By knocking out or inhibiting the GhHB6 gene in cotton using gene editing technologies such as CRISPR-Cas9, new cotton varieties with superior fiber quality can be bred. These new varieties have fibers that are longer, more uniform, and stronger, aiming to enhance the economic value of cotton and the quality of textiles.

[0153] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments without creative effort, as shown in these embodiments, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Knockout GhHB6 Gene and / or knockout GhHB6 The application of gene-based biomaterials in improving cotton fiber quality, the aforementioned GhHB6 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:1; The improved cotton fiber quality includes any one or more of the following: 1) Increase the length of cotton fibers; 2) Improve the breaking strength of cotton fibers; 3) Improve the uniformity of cotton fibers.

2. The application as described in claim 1, characterized in that, The GhHB6 The nucleotide sequence of the gene is shown in SEQ ID NO:

2.

3. The application as described in claim 1, characterized in that, The knockout GhHB6 Biological materials for genes include sgRNA, knockout GhHB6 Recombinant expression vectors and knockout of genes GhHB6 Microorganisms that regenerate genes.

4. The application as described in claim 3, characterized in that, The sequence of the sgRNA is shown in SEQ ID NO:

3.

5. The application as described in claim 3, characterized in that, The knockout GhHB6 The recombinant expression vector of the gene includes a backbone vector; the backbone vector includes the pRGEB32-GhU6.7 vector.

6. The application as described in claim 3, characterized in that, The knockout GhHB6 The recombinant microorganisms of the gene include basic microorganisms; said basic microorganisms include Agrobacterium EHA105.

7. A method for improving the quality of cotton fibers, characterized in that the steps include... include: Knock out the target plant GhHB6 Genes that result in plants with improved cotton fiber quality; The GhHB6 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:1; The improved cotton fiber quality includes any one or more of the following: 1) Cotton fiber length elongation; 2) The breaking strength of cotton fibers is improved; 3) Improved uniformity of cotton fibers.

8. The method as described in claim 7, characterized in that, The cotton includes upland cotton.

9. A genetically modified cotton, characterized in that, The cotton genome GhHB6 The gene was knocked out; GhHB6 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:1.

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