Cotton NAC transcription factor GhCBNAC gene and application thereof

Abstract

The invention belongs to the technical field of cotton gene engineering, and particularly relates to a cotton NAC transcription factor GhCBNAC gene and application thereof. The invention provides application of a GhCBNAC gene knockout and / or a biological material with the GhCBNAC gene knockout in improvement of cotton fiber quality. The GhCBNAC gene encodes an amino acid sequence as shown in SEQ ID NO: 1. According to the invention, cotton fiber elongation, strength increase and micronaire value reduction can be promoted by knocking out the GhCBNAC gene in cotton. Therefore, the application of the GhCBNAC gene provided by the invention provides an effective way for improving the cotton fiber quality.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of cotton genetic engineering, and particularly relates to a cotton NAC transcription factor GhCBNAC gene and application. BACKGROUND

[0002] Cotton is an important strategic material and industrial raw material, and its yield and quality are crucial. At present, in the field of cotton breeding, relevant research shows that the number of fiber initiation and fiber length are key factors to determine the yield and quality of cotton, and fiber strength, as one of the core physical properties of cotton fiber, is a quality index highly concerned by the textile industry. Stronger fiber can improve yarn toughness and fabric durability, which is crucial for the production of high-end textile products.

[0003] From the perspective of basic research, as a single-cell non-glandular epidermal hair, cotton fiber provides an ideal model for the study of cell fate determination, cell elongation and secondary cell wall construction: cotton fiber development needs to go through four closely related stages of initiation, elongation, secondary cell wall (SCW) thickening and maturation, among which the elongation stage mainly affects fiber length, and the secondary cell wall thickening stage is the core period of determining fiber strength. The cellulose content in the secondary cell wall of mature cotton fiber is more than 90%, and the cellulose microfibril formed by the assembly of beta-1, 4-glucose chains is the main load-bearing structure of the cell wall, and its accumulation density and arrangement order directly determine the mechanical strength of the fiber. Through in-depth research on the key stages of fiber development, both the molecular mechanisms of epidermal cell differentiation into fiber cells and the precise construction of secondary cell walls can be revealed, and the key molecular switches and regulatory networks that regulate these processes can also be explored.

[0004] In the process of cotton fiber development, a small number of studies have shown that NAC transcription factors play a key role in the process of fiber secondary wall thickening. For example, overexpression of GhFSN1 can increase the cell wall thickness of mature fibers. However, there is no related report on NAC transcription factors that can increase the length of cotton fibers. SUMMARY

[0005] In order to solve the problem of insufficient gene resources related to cotton fiber elongation in the prior art, the application provides a cotton NAC transcription factor GhCBNAC gene and application, which specifically comprises the following technical scheme: The application provides a biological material for knocking out GhCBNAC gene and / or knocking out GhCBNAC gene, and application of the biological material in improving the quality of cotton fibers. GhCBNAC The gene encodes an amino acid sequence as shown in SEQ ID NO: 1.

[0006] Preferably, the GhCBNACThe nucleotide sequence of the gene is shown as SEQ ID NO: 2.

[0007] Preferably, the improved cotton fiber quality comprises any one or more of the following: 1) increasing the length of cotton fiber; 2) increasing the elongation of cotton fiber; 3) increasing the breaking specific strength of cotton fiber; 4) increasing the uniformity of cotton fiber; 5) reducing the micronaire of cotton, balancing the fineness and maturity of cotton.

[0008] Preferably, the knockout GhCBNAC gene comprises an sgRNA, a recombinant vector for knocking out GhCBNAC the gene, and a recombinant microorganism for knocking out GhCBNAC the gene.

[0009] Preferably, the sgRNA comprises a sequence shown as SEQ ID NO: 3.

[0010] Preferably, the recombinant vector for knocking out the gene comprises a backbone vector; and the backbone vector comprises a pRGEB32-GhU6.7 vector.

[0011] Preferably, the recombinant microorganism for knocking out GhCBNAC the gene comprises a basic microorganism; and the basic microorganism comprises Agrobacterium.

[0012] The present application also provides a method for improving the quality of cotton fiber, comprising the steps of: knocking out GhCBNAC a gene in a target plant to obtain a plant with improved cotton fiber quality; the GhCBNAC gene encodes an amino acid sequence shown as SEQ ID NO: 1.

[0013] Preferably, the cotton comprises Gossypium hirsutum.

[0014] Preferably, the improved cotton fiber quality comprises any one or more of the following: 1) increasing the length of cotton fiber; 2) increasing the elongation of cotton fiber; 3) increasing the breaking specific strength of cotton fiber; 4) increasing the uniformity of cotton fiber; 5) reducing the micronaire of cotton, balancing the fineness and maturity of cotton.

[0015] The present application has the beneficial effects of: The present application provides a method for knocking out GhCBNAC a gene and / or a method for knocking outGhCBNAC application of the biological material of the gene in improving fiber quality of cotton GhCBNAC The gene encodes an amino acid sequence as shown in SEQ ID NO: 1. The present application finds that by knocking out the gene in cotton, GhCBNAC The gene can promote elongation of cotton fiber, increase strength, and reduce micronaire value. Therefore, the application of the gene provided by the present application provides an effective way to improve fiber quality of cotton. GhCBNAC The application of the gene provides an effective way to improve fiber quality of cotton. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced as follows.

[0017] Figure 1 A map of the pRGEB32-GhU6.7-GhCBNAC mutant plasmid vector provided by the present application; Figure 2 A gel map of the mutant transgenic material Cas9 positive detection result provided by the present application; Figure 3 A gel map of the mutant transgenic material NPTII positive detection result provided by the present application; Figure 4 A gel map of the mutant transgenic material sgRNA positive detection result provided by the present application; Among them, Figures 2-4 Among them, the leftmost lane is Marker electrophoresis result, from top to bottom, 100 bp, 250 bp, 500 bp, 750 bp, 1000 bp, 2000 bp, 3000 bp and 5000 bp, CR-1 and CR-2 represent GhCBNAC knockout lines, N represents wild type receptor material, as WT, P represents positive plasmid, as positive control; Figure 5 A Hi-TOM detection result map of the mutant transgenic material provided by the present application; Figure 6 A fiber hand comb result map of the transgenic material provided by the present application; Among them, the scale information of each small graph is 1 cm; Figure 7 A fiber length column chart of the mutant at different periods; Among them, the horizontal coordinates represent 8 DPA, 12 DPA, 16 DPA, 20 DPA and mature period (Mature) in turn. DETAILED DESCRIPTION

[0018] The present application provides a knockout GhCBNAC The gene and / or knockout GhCBNACBiological material of the gene in improving the fiber quality of cotton, the gene GhCBNAC The gene encodes an amino acid sequence as shown in SEQ ID NO: 1.

[0019] SEQ ID NO: 1: MEVLTMETLPLGFRFRPTDEELINHYLRLKINGRHSEVEVIPEIDVCKWEPWDLPGLSVIKSDDPEWFFFCPRDRKYPNGHRSNRATDKGYWKATGKDRTIKSKKSLIGMKKTLVFYKGRAPKGERTNWIMHEYRPTTRELDGTAPGQSAFVLCRLFHKVEGRNDIVKYDEVEQTGYSPAMIKSSPDDTSSDLLQDTVSSDTQAQKPDNLMQNDQITGDTSSCNSHMTSNADYHATEETMVEKYPLLGSNSNLYEPNYGEIDSKVFSPLMNSQLFEDLSFFVDSPYANDFGHDQNGFHFQDGTSEQDVSFPFLDDILSNHYDSCEESNTQKNLVDGTEMPLFGDSFISKPPPPEISYLKENGRPADTDTEMPKLQFGTEVGARRWLGGPIDNNQSLQMQTSFEPTHTQPAFYNQEYRTRNISGLGNYSVGQGTFTDSAMGNINNLQQLTSLKIHVNSGGDLGGDIGMKTGTCQPLKQLNSENFGTRGAGIKIRTRGSQQQPNSDIVNQGTAPRRICLMKLSSGPMKGSVGCVDDGKMMSTGLVEEEEVQSALTEVTEAEAAEQTSSSDESEKNIVNQVTAPRRIHLQTKLSTGAMKGFTGCVNGGNMTSPGLVEEEVQSALTEVTEAEAAGLTSSSDELEKNIVNQGTAPRRISLQINLSTGPMKGSAGCVDGGSMTSPGLVEEEVQSALTEVTEAEAVGQTSSSDESEMNIVNQAIAPRRIRLQLSTGPMNGSAGCIDGGNMTSPGLGKEEVQSALTEATEDEATGQISSSCKTSSSDESEVENRFLKFEGSRDIADESYSKPRLRVKQVDEKHSCSEKGPSLHLKPAPALHRPNSLLVPGIAIFTITLLFALFMGIWL.

[0020] As an embodiment, theGhCBNAC The nucleotide sequence of the gene is set forth in SEQ ID NO: 2.

[0021]

[0022] In one embodiment, the improvement of cotton fiber quality includes any one or more of the following: 1) increasing cotton fiber length; 2) increasing cotton fiber elongation; 3) increasing cotton fiber breaking strength; 4) improving cotton fiber uniformity; 5) reducing cotton micronaire value and balancing cotton fineness and maturity. In one embodiment, increasing cotton fiber length includes increasing the length of the upper half of the cotton fiber.

[0023] As one implementation method, the knockout GhCBNAC Biological materials for genes include sgRNA, knockout GhCBNAC Gene recombination vectors and knockout GhCBNAC Recombinant microorganisms of genes. As one embodiment, the sgRNA comprises the sequence shown in SEQ ID NO:3. As one embodiment, the knockout... GhCBNAC The gene recombination vector includes a backbone vector. As one embodiment, the backbone vector includes a plasmid vector. As one embodiment, the plasmid vector includes the pRGEB32-GhU6.7 vector. As one embodiment, the knockout... GhCBNAC The recombinant microorganisms of the gene include basic microorganisms. In one embodiment, the basic microorganisms include bacteria. In one embodiment, the bacteria include Agrobacterium. In one embodiment, the Agrobacterium preferably includes Agrobacterium EHA105.

[0024] The present invention also provides a method for improving the quality of cotton fibers, comprising the following steps: Knockout of target plants GhCBNAC The gene was used to obtain plants with improved cotton fiber quality; the GhCBNAC gene encodes the amino acid sequence shown in SEQ ID NO:1.

[0025] In one embodiment, the cotton includes upland cotton. In one embodiment, the improvement of cotton fiber quality includes any one or more of the following: 1) increasing cotton fiber length; 2) increasing cotton fiber elongation; 3) increasing cotton fiber breaking strength; 4) improving cotton fiber uniformity; 5) reducing cotton micronaire value and balancing cotton fineness and maturity. In one embodiment, increasing cotton fiber length includes increasing the length of the upper half of the cotton fiber.

[0026] To further illustrate the present invention, the following description, in conjunction with the accompanying drawings and embodiments, describes a cotton NAC transcription factor provided by the present invention. GhCBNAC The genes and their applications are described in detail, but they should not be construed as limiting the scope of protection of this invention.

[0027] The reference source of the cotton genome sequence relied on by the present application is: Wang et al. Reference genome sequences of two cultivated allotetraploid cottons, Gossypium hirsutum and Gossypium barbadense. Nat Genet. 2019 Feb;51(2):224-229. doi: 10.1038 / s41588-018-0282-x. Epub 2018 Dec 3. PMID: 30510239. Example 1 Construction of GhCBNAC CRISPR vector The target was searched for reference CRISPR-P website http: / / crispr.hzau.edu.cn / cgi-bin / CRISPR2 / CRISPR, and an sgRNA sequence with a higher score located in the exon region was selected, as shown in SEQ ID NO: 3. The primers used for vector construction were designed according to the sgRNA sequence, as follows: sgRNA (SEQ ID NO: 3): 5'-CGTGACAGGAAGTACCCCAA-3'; pRGEB32-GhU6.7-F (SEQ ID NO: 4): 5'-AAGCATCAGATGGGCAAACAAAGCACCAGTGGTCTAG-3'; CR-CBNAC-R (SEQ ID NO: 5): 5'-TTCTAGCTCTAAAACTTGGGGTACTTCCTGTCACGTGCACCAGCCGGGAAT-3'.

[0028] This example is to recombine a single sgRNA sequence into a pRGEB32-GhU6.7 vector. The primers pRGEB32-GhU6.7-F and CR-CBNAC-R are used for PCR product amplification to obtain a PCR product containing trRNA-gRNA. The PCR product and the linearized expression vector are connected by In-fusion using Exnase enzyme, and the completed vector is named pRGEB32-GhU6.7-GhCBNAC. The map of the vector is shown in Bsa Ⅰ enzyme is used to cut the pRGEB32-GhU6.7 empty vector at 37°C for 6 h, and gel electrophoresis is performed after cutting. The large fragment of the pRGEB32-GhU6.7 empty vector is recovered by gel excision, and the PCR product and the linearized expression vector are connected by In-fusion using Exnase enzyme, and the completed vector is named pRGEB32-GhU6.7-GhCBNAC. The map of the vector is shown in Figure 1 , Figure 1The vector backbone is pRGEB32-GhU6.7, which is used to provide kanamycin resistance in bacteria, and the transgenic plants are also kanamycin resistant.

[0029] The reaction product was used to transform *E. coli* competent cells TOP10. After 10–12 h, single clones were picked for PCR positive detection. The primer sequences used for PCR are shown below: U6-7S (SEQ ID NO:6): 5′-TGTGCCACTCCAAAGACATCAG-3′; CR-CBNAC-R (SEQ ID NO: 7): 5′-TTCTAGCTCTAAAACTTGGGGTACTTCCTGTCACGTGCACCAGCCGGGAAT-3′.

[0030] The PCR reaction system consisted of 20 μL of: 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.

[0031] The PCR reaction conditions were as follows: 95 °C pre-denaturation for 5 min; 95 °C for 30 sec, 58 °C for 30 sec, 72 °C for 20 sec, 28 cycles; 72 °C extension for 5 min.

[0032] The positive monoclonal clones were propagated and the plasmids were extracted to obtain the mutant plasmid pRGEB32-GhU6.7-GhCBNAC for transformation.

[0033] Example 2 GhCBNAC Genetic transformation of knockout vectors and screening and identification of knockout lines A. Agrobacterium-mediated genetic transformation 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.

[0034] The glycerol tube containing the EHA105 strain with the target gene (i.e. the pRGEB32-GhU6.7-GhCBNAC vector constructed in the application) stored in the -80°C refrigerator was taken out, melted on ice, and 10 μL was inoculated into 2 mL of LB liquid containing 100 mg / L kanamycin and cultured at 28°C for 1 day. 20 μL of the activated bacterial liquid was inoculated into 20 mL of fresh liquid LB containing 100 mg / L kanamycin and cultured at 28°C overnight. 1 mL of the turbid bacterial liquid was taken and centrifuged at 12000 rpm for 30 s to collect the bacterial cells, which were resuspended in 20 mL of MGL medium containing 50 mg / L acetyl-syringone (AS) and cultured at 28°C for 30 min, and used to infect the hypocotyls.

[0035] The specific steps of Agrobacterium-mediated transformation of cotton hypocotyls are as follows: (1) In the clean bench, 30 sterile seedlings were taken, and the hypocotyls were cut into 0.5-0.8 cm segments on sterile filter paper and inoculated into a 50 mL sterile conical flask containing the activated EHA105 Agrobacterium liquid containing the target vector pRGEB32-GhU6.7-GhCBNAC, and the infection was performed for 5 min with shaking several times; (2) The bacterial liquid was poured out, the hypocotyls were placed on sterile filter paper to absorb the surface bacterial liquid, and then placed in the clean bench for 10-15 min and inoculated into 2,4-D induction medium without antibiotics, and cultured at 19°C in the dark for 48 h; (3) After the co-culture, the hypocotyls were cut and inoculated into 2,4-D induction medium containing kanamycin (100 mg / L) and cephalosporin (100 mg / L), and cultured at 28°C under weak light (cool light source 135 μmol m -2 s -1 ) for continuous subculture until embryogenic calli appeared; (4) The embryogenic calli were successively inoculated into embryo differentiation medium for subculture until somatic embryos matured, and the matured cotyledon embryos were inoculated into rooting medium for germination until complete plants were obtained.

[0036] The culture formula used in this example is as follows: 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 was added to 1 L.

[0037] 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).

[0038] 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.

[0039] 2,4-D induction medium: MS medium was used as the basal medium, with the addition of 0.1 mg / L 2,4-D, 0.1 mg / L cytokinin (KT), 30 g / L glucose, and 2.5 g / L Phytagel, and the volume was supplemented with distilled water to 1 L. The pH was adjusted to 5.9.

[0040] 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.

[0041] Rooting medium: Use 1 / 2 MS 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.

[0042] B. Identification of transgenic plants (1) Positive detection of transgenic T0 generation plants Genomic DNA was extracted from the young leaves of the transgenic plants. DNA extraction was performed using the Plant Genomic DNA Extraction Kit from Tiangen Biotech (Beijing) Technology Co., Ltd. Specific operating procedures are detailed in the kit's instructions. The primers for PCR amplification of Cas9-positive transgenic materials are shown below: Cas9-F (SEQ ID NO: 8): 5'-GCTTGTGCGTTTCGATTTGA-3'; Cas9-R (SEQ ID NO: 9): 5'-CCGCTCGTGCTTCTTATCCT-3'.

[0043] The length of the target gene fragment is 999 bp.

[0044] The primers for PCR amplification for positive detection of the transgenic material NPTII are as follows: NPTII-F (SEQ ID NO: 10): 5'-GCTTGGGTGGAGAGGCTATTC-3'; NPTII-R (SEQ ID NO: 11): 5'-GAAGAACTCGTCAAGAAGGCG-3'.

[0045] Since the vector contains the NPTII resistance gene, if the band can be detected, it means that the plant is a positive plant.

[0046] The length of the target gene fragment is 750 bp.

[0047] The primers for PCR amplification for positive detection of the transgenic material sgRNA are as follows: sgRNA-F (SEQ ID NO: 12): 5'-TATAAGCGAAAGAAGCATCAGA-3'; sgRNA-R (SEQ ID NO: 13): 5'-GACCCGAATTTGTGGACC-3'.

[0048] The length of the target gene fragment is 290 bp.

[0049] PCR detection of whether there is corresponding T-DNA insertion is carried out using the three pairs of primers described above, and the use of multiple pairs of primers can further ensure the accuracy of the experiment. The system of PCR reaction is 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; DNA 1 μL. The PCR reaction conditions are: 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. The positive detection results of the transgenic plants are shown in Figure 2 , Figure 3 and Figure 4 .

[0050] Example 3 GhCBNAC CRISPR knockout line editing efficiency detection The method for extracting cotton leaf DNA is shown in Example 2. Since the CRISPR-Cas9 technology can cause a phenomenon of multiple editing types on a single plant, and sanger sequencing cannot identify multiple mutation types, high-throughput Hi-TOM sequencing technology is used to detect the editing efficiency of knockout lines. The specific steps are as follows: (1) According to the conventional PCR primer design principle, the primer is designed (16-20 nt); the target point needs to be within 10-100 bp away from the left primer or the right primer. For independent single plant material of gene knockout, site-specific primers with common adapter sequences (SEQ ID NO: 14: 5'-ggagtgagtacggtgtgc-3' and SEQ ID NO: 15: 5'-gagttggatgctggatgg-3') are designed to amplify the genomic sequence of the target gene by first-round PCR.

[0051] The first-round PCR reaction system (20 μL) contains: 50 ng genomic DNA, 0.2 μL 10 µmol / L forward and reverse primers, and 10 μL 2×Taq Master Mix, supplemented with ddH2O to 20 μL.

[0052] The first-round PCR reaction program is: 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.

[0053] (2) The second-round PCR uses the first-round PCR product as a template, and adds primers containing Barcode and Index for PCR amplification.

[0054] The second-round PCR reaction system (20 μL) contains: 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, supplemented with ddH2O to 20 μL.

[0055] The second-round PCR reaction program is: 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.

[0056] (3) The obtained PCR products were mixed in equal amounts, purified using a purification kit (OMEGA, D2500-2), and each 96-well mixed sample was subjected to 1 G data amount on a second-generation sequencing platform. The data were analyzed using a Hi-TOM analysis website (http: / / www.hi-tom.net / hi-tom / ). The determined sequences were compared with the sequences of the reference genome, and the final editing efficiency was evaluated.

[0057] In combination with the Barcode labeling method, each single strain of the knockout material edited was labeled with a Barcode at the 5' end of the forward and reverse primers for detecting the target site in the form of a linker, so that each single strain obtained a specific pair of Barcode labels. The sequences of the target sites of the independent single strains were amplified using these primers containing Barcodes, a mixed DNA library was constructed, and high-throughput sequencing was performed. The sequencing results were sorted according to the Barcode-labeled primers, and the corresponding sequencing results of the independent single strains were obtained. The sequencing results were subjected to removal of repeated and low-quality sequences, and then compared and analyzed with the reference gene sequence, thereby completing the process of detecting the mutation of the target gene site of the single strain.

[0058] The Barcode primers used in the detection were as follows: F-1 (SEQ ID NO: 16): 5'-GCTTGCGTTGGAGTGAGTACGGTGTGC-3'; F-2 (SEQ ID NO: 17): 5'-GCTTGTAGTGGAGTGAGTACGGTGTGC-3'; F-3 (SEQ ID NO: 18): 5'-GCTTACGCTGGAGTGAGTACGGTGTGC-3'; F-4 (SEQ ID NO: 19): 5'-GCTTCTCGTGGAGTGAGTACGGTGTGC-3'; F-5 (SEQ ID NO: 20): 5'-GCTTGCTCTGGAGTGAGTACGGTGTGC-3'; F-6 (SEQ ID NO: 21): 5'-GCTTAGTCTGGAGTGAGTACGGTGTGC-3'; F-7 (SEQ ID NO: 22): 5'-GCTTCGACTGGAGTGAGTACGGTGTGC-3'; F-8 (SEQ ID NO: 23): 5'-GCTTGATGTGGAGTGAGTACGGTGTGC-3'; F-9 (SEQ ID NO: 24): 5'-GCTTATACTGGAGTGAGTACGGTGTGC-3'; F-10 (SEQ ID NO: 25): 5'-GCTTCACATGGAGTGAGTACGGTGTGC-3'; F-11 (SEQ ID NO: 26): 5'-GCTTGTGCTGGAGTGAGTACGGTGTGC-3'; F-12 (SEQ ID NO: 27): 5'-GCTTACTATGGAGTGAGTACGGTGTGC-3'; R-A (SEQ ID NO: 28): 5'-CCATCCAGCATCCAACTCAACGCACAG-3'; R-B (SEQ ID NO: 29): 5'-CCATCCAGCATCCAACTCACTACACAG-3'; R-C (SEQ ID NO: 30): 5'-CCATCCAGCATCCAACTCAGCGTACAG-3'; R-D (SEQ ID NO: 31): 5'-CCATCCAGCATCCAACTCACGAGACAG-3'; R-E (SEQ ID NO: 32): 5'-CCATCCAGCATCCAACTCAGAGCACAG-3'; R-F (SEQ ID NO: 33): 5'-CCATCCAGCATCCAACTCAGACTACAG-3'; R-G (SEQ ID NO: 34): 5'-CCATCCAGCATCCAACTCAGTCGACAG-3'; R-H (SEQ ID NO: 35): 5'-CCATCCAGCATCCAACTCACATCACAG-3'.

[0059] The Index primers used for detection are: 2P-F-1 (SEQ ID NO: 36): 5'-AATGATACGGCGACCACCGAGATCTACACAGGAACCTACACTCTTTCCCTACACGACGCTCTT-3'; 2P-R-1 (SEQ ID NO: 37): 5′-CAAGCAGAAGACGGCATACGAGATCTTAGCCAGTGACTGGAGTTCAGACGTGTGCTCTT-3′.

[0060] The PCR reaction conditions were as follows: 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.

[0061] The PCR reaction system consisted of: 1 μL of the first-round PCR product, 0.4 nmol / L of forward and reverse primers, 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 volume to 20 μL.

[0062] Equal volumes of PCR product were taken, purified, and then sequenced. The results are as follows: Figure 5 As shown, this invention has successfully constructed a transgenic strain.

[0063] Example 4: Using genetically modified cotton to... GhCBNAC Gene functional verification A. GhCBNAC Measurement of immature fiber length in transgenic lines In the experimental field of Huazhong Agricultural University, cotton bolls from the same part of the transgenic and control lines were harvested at the same time point for fiber length measurement. Each line had 20 biological replicates, and the cotton bolls were 8 DPA, 12 DPA, 16 DPA and 20 DPA.

[0064] The ovules from the same part of the cotton boll were placed in boiling water and gently tapped with a glass rod to disperse the fibers. They were then rinsed under running water to straighten the fibers, and finally stretched on a clean table for length measurement. The obtained data were analyzed using Prism software for multiple comparisons, and the results are shown below. Figure 6 It is evident that, compared to the wild type, the knockout... GhCBNAC Cotton fiber elongation can be significantly promoted at 8 DPA, 12 DPA, 16 DPA and 20 DPA.

[0065] B. GhCBNAC Determination of mature fiber length in knockout lines Will GhCBNACThe knockout strains CR-1, CR-2, CR-3 and wild type material (WT) are planted in a transgenic test field, 30 plants for each strain, and when the cotton fibers are mature, the cotton bolls of the same part of the transgenic and control strains are picked at the same time point for fiber length measurement, 20 biological repeats for each strain, and the cotton seed carding method is used for measurement. First, straighten the fibers along the middle ventral groove of the cotton seed, and then card the fibers with a comb. Then, measure the cotton seed ventral groove downward on the black wool board with a steel ruler. The obtained data is analyzed by prism software for multiple comparison analysis, and the results are shown in Figure 6 and Figure 7 Compared with the wild type, the knockout GhCBNAC can significantly promote the elongation of cotton fibers Table 1: Hand carding statistical results of fiber length of transgenic materials

[0066] In Table 1, different lowercase letters represent significant differences, P <0.05.

[0067] C. GhCBNAC Mature fiber quality identification of knockout strains The mature cotton bolls in the middle of the plant at the same period are picked by hand and are machine ginned. The weight of each fiber sample is about 10 g. The arranged samples are measured for five indicators, and the instrument used is a high volume cotton fiber tester (High Volume Instrument, HFT9000, Premier, India). Each transgenic strain has at least three repeats. The obtained data is analyzed by prism software for multiple comparison analysis. The results are shown in Table 2, and it can be seen that compared with the wild type, the knockout GhCBNAC gene can significantly promote the elongation of cotton fibers, increase the strength and reduce the micronaire value.

[0068] Table 2: Mature fiber quality determination results of transgenic materials

[0069] In Table 2, different lowercase letters represent significant differences, P <0.05; T4 represents the generation of transgenic cotton.

[0070] In summary, the present application finds that by knocking out the GhCBNAC gene in cotton, the elongation of cotton fibers is promoted, the strength is increased, and the micronaire value is reduced. Therefore, the application of the GhCBNAC GhCBNAC gene provided by the present application provides an effective way to improve the fiber quality of cotton.

[0071] Although the above embodiments have been described in detail, it should be understood that these are only some embodiments of the present application, but not all embodiments. Other embodiments can be obtained without creativity based on the embodiments, and these embodiments also belong to the protection scope of the present application.

Claims

1. Knockout GhCBNAC Gene and / or knockout GhCBNAC The application of gene-based biomaterials in improving cotton fiber quality, the aforementioned GhCBNAC The gene encodes the amino acid sequence shown in SEQ ID NO:

1.

2. The application as described in claim 1, characterized in that, The GhCBNAC 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 improved cotton fiber quality includes any one or more of the following: 1) Increase the length of cotton fibers; 2) Improve cotton fiber elongation; 3) Improve the breaking strength of cotton fibers; 4) Improve the uniformity of cotton fibers; 5) Reduce the micronaire value of cotton to balance the fineness and maturity of cotton.

4. The application as described in claim 1, characterized in that, The knockout GhCBNAC Biological materials for genes include sgRNA, knockout GhCBNAC Gene recombination vectors and knockout GhCBNAC Microorganisms that regenerate genes.

5. The application as described in claim 4, characterized in that, The sgRNA includes the sequence shown in SEQ ID NO:

3.

6. The application as described in claim 4, characterized in that, The recombinant vector for the knockout gene includes a backbone vector; the backbone vector includes the pRGEB32-GhU6.7 vector.

7. The application as described in claim 4, characterized in that, The knockout GhCBNAC Recombinant microorganisms include basic microorganisms; said basic microorganisms include Agrobacterium.

8. A method for improving the quality of cotton fibers, characterized in that the steps include... include: Knockout of target plants GhCBNAC Genes that result in plants with improved cotton fiber quality; The GhCBNAC The gene encodes the amino acid sequence shown in SEQ ID NO:

1.

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

10. The method as described in claim 8, characterized in that, The improved cotton fiber quality includes any one or more of the following: 1) Increase the length of cotton fibers; 2) Improve cotton fiber elongation; 3) Improve the breaking strength of cotton fibers; 4) Improve the uniformity of cotton fibers; 5) Reduce the micronaire value of cotton to balance the fineness and maturity of cotton.

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