Application of Ghbzip208 gene in promoting elongation of cotton fiber

Abstract

This invention provides GhbZIP208 The application of genes in promoting cotton fiber elongation falls under the field of plant molecular biology. This invention provides a method for regulating... GhbZIP208 The application of gene expression reagents in regulating cotton fiber quality and yield, and the experimental findings on the effects of gene editing on the described [results]. GhbZIP208 Gene expression levels were significantly reduced, and the fiber length of the overexpressing plants was longer than that of the WT plants; while in the overexpressing plants... GhbZIP208 Gene expression levels were significantly increased, and the fiber content of the plants was lower than that of the WT plants; there were significant differences among the overexpressing plants, wild-type plants, and gene-edited plants, indicating that... GhbZIP208 The above plays a negative regulatory role in cotton fiber elongation. GhbZIP208 Genes can be used for genetic improvement or molecular breeding to increase cotton fiber yield, such as breeding high-quality cotton varieties, especially long-fiber cotton varieties.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology technology, specifically relating to a... GhbZIP208 Application of genes in promoting cotton fiber elongation. Background Technology

[0002] Cotton is not only a core raw material in the textile industry, directly supporting my country's position as the world's largest producer and exporter of textiles. Cotton production is mainly based on upland cotton, and the quality of fibers, such as fiber length, strength, and micronaire value, varies considerably and is not very stable across regions. Therefore, increasing cotton yield per unit area, improving fiber quality, and breeding more advantageous cotton varieties are important goals for cotton breeding in my country today.

[0003] bZIP proteins are a class of crucial transcriptional regulators for plant development. The gene family contains multiple members that control various processes of plant growth and development. In Arabidopsis thaliana, 78 members of the bZIP family, AtbZIP1–AtbZIP78, have been identified. They are divided into 13 groups: A, B, C, D, E, F, G, H, I, J, K, M, and S. AtbZIP1 and AtbZIP53 It synergistically regulates metabolic networks such as amino acid breakdown, gluconeogenesis, and energy homeostasis, helping plants survive when resources are scarce and resume growth after stress is relieved. AtbZIP53 Seed maturation is regulated by influencing heterodimerization and protein complex formation. AtbZIP56 It promotes photomorphogenesis by binding to the G-box present in the promoter. Group A has 7 members. AtbZIP35 , AtbZIP36 , AtbZIP37 , AtbZIP38 , AtbZIP39 , AtbZIP40 and AtbZIP66 It is a core factor in response to abscisic acid (ABA) or stress. Currently, there are no reported studies on the function of cotton GhbZIP208 protein in regulating cotton fiber elongation. Summary of the Invention

[0004] This invention provides GhbZIP208 The application of genes in promoting cotton fiber elongation, the GhbZIP208 The gene is related to cotton fiber elongation and can negatively regulate cotton fiber length.

[0005] This invention provides a regulation GhbZIP208 Application of gene expression reagents in regulating cotton fiber quality and yield.

[0006] In one specific embodiment of the present invention, the cotton fiber quality and yield include the length of the cotton fiber.

[0007] In one specific embodiment of the present invention, the regulation includes downregulating or knocking out the GhbZIP208 Genes that promote the elongation of cotton fibers.

[0008] In one specific embodiment of the present invention, the... GhbZIP208 The amino acid sequence of the gene-encoded protein is shown in SEQ ID No. 1.

[0009] The present invention also provides a method for increasing cotton fiber length, comprising downregulating or knocking out [certain substances] in the target cotton genome. GhbZIP208 Gene.

[0010] In one specific embodiment of the present invention, the... GhbZIP208 The nucleotide sequence of the gene is shown in SEQ ID No. 2.

[0011] This invention also provides a method for breeding long-fiber cotton varieties, including downregulation or knockout in the target cotton genome. GhbZIP208 Gene.

[0012] In one specific embodiment of the present invention, the silence GhbZIP208 Genetic methods, including constructing GhbZIP208 The gene knockout vector was transformed into the target cotton genome using Agrobacterium-mediated genetic transformation. Construct the GhbZIP208 The primer pairs for the gene knockout vector include upstream primers with nucleotide sequences as shown in SEQ ID No. 13 and SEQ ID No. 14, and downstream primers as shown in SEQ ID No. 15 and SEQ ID No. 16.

[0013] This invention also provides a method for breeding long-fiber cotton varieties, which involves analyzing the cotton genome... GhbZIP208 Gene expression levels were detected, and varieties with lower expression levels were cotton varieties with longer fibers.

[0014] In one specific embodiment of the present invention, the detection of the GhbZIP208 Primer pairs for gene expression, including an upstream primer with nucleotide sequences as shown in SEQ ID No. 5 and a downstream primer as shown in SEQ ID No. 6.

[0015] Beneficial effects: This invention discovered that the GhbZIP208 gene is specifically expressed in fiber ovules. In DP5690 and its two short-fiber mutants, Li1_DP5690 and Li2_DP5690, the expression level of the wild-type DP5690 is lower than that of the short-fiber micro-mutant. Between Sea Island cotton and Upland cotton, which have different fiber qualities, Upland cotton... GhbZIP208Lower expression levels and longer fiber lengths were observed. In this embodiment of the invention, gene-editing vectors and overexpression vectors were constructed, and gene-edited and overexpression plants were cultured after genetic transformation. The results showed that the gene-edited plants exhibited the characteristics described... GhbZIP208 Gene expression levels were significantly reduced, and the fiber length of the overexpressing plants was longer than that of the WT plants; while in the overexpressing plants... GhbZIP208 Gene expression levels were significantly increased, and the plant fibers were shorter than those of WT plants; there were significant differences among the overexpressing plants, wild-type plants, and gene-edited plants, indicating that... GhbZIP208 The above plays a negative regulatory role in cotton fiber elongation. GhbZIP208 Genes can be used for genetic improvement or molecular breeding to increase cotton fiber yield, such as breeding high-quality cotton varieties, especially long-fiber cotton varieties. Attached Figure Description

[0016] Figure 1 A gene evolutionary tree of the bZIP family; Figure 2 This is a schematic diagram of the bZIP family gene structure analysis; A, B, C, D, E, F, G, H, I, J, K, M, and S in the diagram all represent subgroups. Figure 3 for bZIP208 Statistical graph of expression levels at different fiber development stages. Figure a: Expression level of GhbZIP208 in DP5690, Li1_DP5690 and Li2_DP5690 at 8 DPA of flowering; b: Expression level of GhbZIP208 in Sea Island cotton and Upland cotton at different fiber development stages. Figure 4 For genes GhbZIP208 Subcellular localization results; Figure 5 for GhbZIP208 The identification results of the mutant strain are shown in the figure; Figure 6 For genes GhbZIP208 Figure 1 shows the identification results of the overexpression lines; a: agarose gel electrophoresis results; b: relative expression level. Figure 7 The results are shown in the figure (a) and length statistics of mature fibers of wild-type, mutant and transgenic cotton. Detailed Implementation

[0017] This invention provides a regulation GhbZIP208 Application of gene expression reagents in regulating cotton fiber quality and yield.

[0018] The present invention GhbZIP208The amino acid sequence of the gene-encoded protein is shown in SEQ ID No. 1, and also includes amino acids that maintain a similar function to SEQ ID No. 1 after modification, deletion, substitution, or addition of one or more amino acids based on the sequence shown in SEQ ID No. 1. This invention... GhbZIP208 The gene has a nucleotide sequence as shown in SEQ ID No. 2, or a nucleotide sequence that is more than 90% identical to the nucleotide sequence shown in SEQ ID No. 2 and has the same function.

[0019] This invention, through database analysis and experiments, discovered that the... GhbZIP208 The gene has a special function in the process of cotton fiber elongation; knocking out the gene... GhbZIP208 The cotton plants with the gene have a significantly increased fiber length. Therefore, all those that can reduce the aforementioned GhbZIP208 Reagents for gene expression are all within the scope of protection of this invention, such as those described above. GhbZIP208 Gene expression cassettes, knockout vectors, microorganisms, and other materials containing the gene. GhbZIP208 Genetically modified plant cells, tissues, or plants.

[0020] The cotton fiber quality and yield described in this invention include the length of the cotton fibers.

[0021] The present invention also provides a method for increasing cotton fiber length, comprising downregulating or knocking out [certain substances] in the target cotton genome. GhbZIP208 Gene.

[0022] This invention does not specifically limit the method of overexpression; in one embodiment, genetic transformation is used to transform the overexpression method. GhbZIP208 The gene knockout vector was transformed into the target cotton genome, thereby constructing the desired gene knockout vector. GhbZIP208 The cotton fibers of gene-silenced plants showed a significant increase compared to wild-type gene-knockout plants. The base vectors for the knockout vector described in this invention include pKSE401 and pCBC-DT1T2.

[0023] This invention also provides a method for breeding long-fiber cotton varieties, including downregulation or knockout in the target cotton genome. GhbZIP208 Gene.

[0024] The silence described in this invention GhbZIP208 Genetic methods, including constructing GhbZIP208 A gene knockout vector was transformed into the target cotton genome using an Agrobacterium-mediated genetic transformation method. Construct the GhbZIP208The primer pairs for the gene knockout vector include upstream primers with nucleotide sequences as shown in SEQ ID No. 13 and SEQ ID No. 14, and downstream primers as shown in SEQ ID No. 15 and SEQ ID No. 16.

[0025] DT1-BsF (SEQ ID No. 13): 5'-TATATGGTCTCGATTGNNNNNNNNNNNNNNNNNNNGTT-3'; DT1-F0 (SEQ ID No. 14): 5'-TGNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGC-3'; DT2-R0 (SEQ ID No. 15): 5'-AACNNNNNNNNNNNNNNNNNCAATCTCTTAGTCGACTCTAC-3'; DT2-BsR (SEQ ID No. 16): 5'-ATTATTGGTCTCGAAACNNNNNNNNNNNNNNNNNCAA-3'.

[0026] This invention also provides a method for breeding long-fiber cotton varieties, which involves analyzing the cotton genome... GhbZIP208 Gene expression levels were detected, and varieties with lower expression levels were cotton varieties with longer fibers.

[0027] The present invention detects the GhbZIP208 Primer pairs for gene expression levels, including an upstream primer with nucleotide sequences as shown in SEQ ID No. 5 and a downstream primer as shown in SEQ ID No. 6; in one embodiment, using... GhUBQ7 The internal reference gene was designed, and the upstream primer shown in SEQ ID No. 7 and the downstream primer shown in SEQ ID No. 8 were designed.

[0028] qPCR-bZIP208-F (SEQ ID No.5): 5'-GAAGCCTGATTCAAAGCTTGTTT-3'; qPCR-bZIP208-R (SEQ ID No. 6): 5'-TGGGTGATAATCTGTGGGAGAAA-3'; GhUBQ7-F (SEQ ID No. 7): 5'-GAAGGCATCCACCTGACCAAC-3'; GhUBQ7-R (SEQ ID No. 8): 5'-CTTGACCTTCTTCTTCTTGTGCTTG-3'; To further illustrate the present invention, the following description, in conjunction with embodiments, provides a method for implementing the present invention. GhbZIP208 The application of genes in promoting cotton fiber elongation is described in detail, but it should not be construed as limiting the scope of protection of this invention.

[0029] Unless otherwise specified, the materials and reagents used in the embodiments of this invention are prepared according to existing methods or purchased directly from the market. The following is a description of some of the experimental materials used in the embodiments of this invention: The transgenic cotton recipient material used in this embodiment is upland cotton (Gossypium hirsutum) "Xinluzhong 75" (New Cotton Approval No. 64, 2014).

[0030] The vectors used in this embodiment are as follows: pKSE401: a CRISPR-Cas9-encoded plant expression vector used for the transformation of mutant materials; pCambia2300-GFP: an overexpression vector used for the transformation of overexpression materials; and pSGFP: a subcellular localization vector used for transient transformation of tobacco.

[0031] The strains used in this embodiment are as follows: Escherichia coli competent cells DH5α and Agrobacterium competent cells GV3101.

[0032] Example 1 GhbZIP Evolutionary analysis of genes Downloaded upland cotton and Arabidopsis data were used to establish a local database using BLAST. The amino acid sequences of Arabidopsis bZIP were used as query sequences, and BLASTP alignment was employed to obtain candidate bZIP genes in upland cotton. Then, 78 Arabidopsis bZIP amino acid sequences were submitted to the online Pfam database for analysis to identify conserved domains of Arabidopsis bZIP. The online Pfam database was used to determine whether candidate sequences contained conserved domains such as bZIP_1, bZIP_2, and bZIP_plant_GBF1, thereby obtaining the cotton bZIP family genes.

[0033] Using the amino acid sequence of the upland cotton bZIP gene, a phylogenetic tree was constructed using muscle and fasttree. Based on the genome annotation file obtained from the cottongen website, the Visualize Gene Structure tool in TBtools was used to analyze and visualize the cotton gene structure. Annotation information for the cotton bZIP gene family was compiled into a gff3 file, and the start positions and key structures of all full-length cotton bZIP genes were compiled into a text file. These compiled files were imported into TBtools, and the software was run to obtain the gene structure diagram.

[0034] Ultimately, 221 bZIP genes were identified in upland cotton. Phylogenetic analysis of the bZIP genes in Arabidopsis and upland cotton revealed that these bZIP genes were divided into thirteen subgroups (…). Figure 1 By identifying the domains specific to each gene subgroup using NCBI, it was found that subgroup A contains domains such as bZIP_plant_BZIP46, subgroup B contains domains such as bZIP_HY5-like, subgroup C contains domains such as bZIP_plant_GBF1 and bZIP_C, and subgroup D contains domains such as bZIP_HBP1b-like, and most of them also contain the DOG1 domain. Subgroup E contains domains such as bZIP_plant_RF2; subgroup F contains domains such as bZIP_2 or BRLZ; subgroup G contains domains such as BRLZ, bZIP_plant_GBF1, and bZIP_1, and also contains the G-box binding domain MFMR; subgroup H contains domains such as bZIP_HY5-like and BRLZ; subgroup I contains domains such as bZIP_plant_RF2, and most also contain mitosis-related domains such as ZapB and MAD; subgroup J contains domains such as bZIP_plant_GBF1; subgroup K contains domains such as bZIP_HY5-like; subgroup M contains domains such as bZIP_plant_RF2; and subgroup S contains domains such as BRLZ and bZIP_plant_GBF1. Figure 2 ).

[0035] Example 2: Transcriptome Analysis of Cotton First, we downloaded expression data related to the GhbZIP208 gene in upland and sea island cotton (Zhao N, Wang W, Grover CE, et al. Genomic and GWAS analyses demonstrate phylogenomicrelations of Gossypium barbadense in China and selection for fiber length, lint percentage and Fusarium wilt resistance), and used expression data related to the GhbZIP208 gene in different varieties (DP5690, Li1_DP5690, Li2_DP5690) from China (Thyssen GN, Fang DD, Turley RB, et al. Next generation genetic mapping of the Ligon-lintless-2 (Li2) locus in upland cotton (Gossypium hirsutum L.)) to analyze the expression level of the GhbZIP208 gene at 8 DPA in DP5690 and its two short fiber mutants Li1_DP5690 and Li2_DP5690, using Graphpad. Pism generates line charts for visual analysis.

[0036] The results are as follows Figure 3 As shown, among the standard systems of Sea Island cotton and Upland cotton with differences in fiber quality, Upland cotton has a lower expression level of GhbZIP208, better fiber quality, and longer fiber length. The expression level of DP5690 wild type is lower than that of short fiber micro mutant.

[0037] Example 3 Construction of gene subcellular localization vector (1) Linearization of expression vector: The editing vector pSGFP (Gao JS, Wu N, Shen ZL, et al. Molecular cloning, expression analysis and subcellular localization of a Transparent Testa 12 ortholog in brown cotton (Gossypium hirsutum L.)) was incubated at 37℃ for 2 h and then digested with a single enzyme to obtain the linearized vector; 50 μL single enzyme digestion system: 2 μg pSGFP vector, 4 μL KpnI, 5 μL rCutSmart and the remainder ddH2O.

[0038] (2) Obtaining the target fragment: Primers carrying the homologous arms of the vector were designed based on the cotton genome sequence, and the target fragment was obtained by PCR amplification. GhbZIP208 Full-length gene. Using cDNA from 15-day-old cotton fiber (cotton fiber tissue 15 days after flowering) of Xinluzhong 75 as a template, the gene with restriction sites was amplified using Novizan's high-fidelity enzyme 2×Phanta Max Master Mix (DyePlus). GhbZIP208 Full-length gene.

[0039] pSGFP-bZIP208-F (SEQ ID No. 9): 5'-atactagtggatccggtaccATGGAAGTGCCTGGATTTGATG-3'; pSGFP-bZIP208-R (SEQ ID No. 10): 5'-cccttgctcaccatggtaccAGAAGCTGATTTAGGTTTCATGGAG-3'; 50 μL amplification system: 25 μL of 2×Phanta Max Master Mix (Dye Plus), 2 μL of template, 2 μL each of forward and reverse primers, and the remainder ddH2O; PCR amplification system: 95 ℃ pre-denaturation for 3 min; 95 ℃ denaturation for 30 s, 58 ℃ annealing for 15 s, 72 ℃ extension for 30 s, 35 cycles; 72 ℃ extension for 5 min.

[0040] (3) Constructing a carrier The linearized vector and the target fragment were homologously recombined using the ClonExpress Ultra One Step Cloning Kit from Nanjing Novizan Biotechnology Co., Ltd. to construct the vector, which was then transformed into Escherichia coli and plated on LB medium containing 10 μg / mL Kan+ to obtain the pSGFP-GhbZIP208 recombinant plasmid.

[0041] The StarPrep Fast Plasmid Mini Kit from Kangrun Company was used to extract plasmids from Escherichia coli. The recombinant plasmids were introduced into Agrobacterium tumefaciens GV3101 to obtain recombinant Agrobacterium. The recombinant Agrobacterium was plated on LB medium containing 10 μg / mL Kan+ and 5 μg / mL Rif+. Single clones were picked and positive strains were detected for transient transformation of tobacco.

[0042] Example 4: Construction of gene editing vector (1) Linearization of expression vector: The editing vector pKSE401 was incubated at 37 °C for 2 h and then digested with a single enzyme to obtain a linearized vector; 50 μL single enzyme digestion system: pKSE401 2 μg, BsaI 4 μL, rCutSmart 5 μL and the remainder ddH2O; (2) sgRNA design: based on GhbZIP208 Two gRNA targets were designed using specific sequences.

[0043] gRNA1-bZIP208 (SEQ ID No. 11): 5'-GATGGTGATCCTCAACCCCA-3'; gRNA2-bZIP208 (SEQ ID No. 12): 5'-TATCCAACAGGGAATC-3'.

[0044] (3) Constructing a carrier PCR amplification was performed using pCBC-DT1T2 (Li X, Liu L, Luo C, Chen Z, Shu B. EfficientCRISPR / Cas9 system established via co-cultivation of plantlets and Agrobacterium tumefaciens for positive transgenic calluses generation and regeneration in cultivated strawberry (Fragaria × ananassa)) diluted 100-fold as a template. The gRNA was ligated to a partial fragment of pCBC-DT1T2, and the enzyme digestion-ligation system was established after gel recovery.

[0045] 50 μL enzyme digestion-ligation system: 2 μL PCR fragment, 2 μL pHSE401, 1.5 μL 10×NEB T4 Buffer, 1.5 μL 10×BSA, 1 μL BsaI (NEB), 1 μL T4 Ligase (NEB), and the remainder ddH2O; Reaction conditions: 37 ℃ for 5 h, 50 ℃ for 5 min, 80 ℃ for 10 min.

[0046] 5 μL of the recombinant plasmid pKSE401-GhbZIP208 was transformed into competent E. coli cells and plated on LB medium containing 10 μg / mL Kan+ to obtain the recombinant plasmid.

[0047] The StarPrep Fast Plasmid Mini Kit from Kangrun Company was used to extract plasmids from Escherichia coli. The recombinant plasmids were introduced into Agrobacterium tumefaciens GV3101 to obtain recombinant Agrobacterium. The recombinant Agrobacterium was plated on LB medium containing 10 μg / mL Kan+ and 5 μg / mL Rif+. Single clones were picked and positive strains were detected for use in cotton genetic transformation.

[0048] DT1-BsF (SEQ ID No. 13): 5'-TATATGGTCTCGATTGNNNNNNNNNNNNNNNNNNNGTT-3'; DT1-F0 (SEQ ID No. 14): 5'-TGNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGC-3'; DT2-R0 (SEQ ID No. 15): 5'-AACNNNNNNNNNNNNNNNNNCAATCTCTTAGTCGACTCTAC-3'; DT2-BsR (SEQ ID No. 16): 5'-ATTATTGGTCTCGAAACNNNNNNNNNNNNNNNNNCAA-3'.

[0049] Example 5: Construction of gene overexpression vector (1) Linearization of expression vector: The overexpression vector pCambia2300-GFP was incubated at 37 °C for 2 h and then double-digested to obtain the linearized vector; 50 μL double enzyme digestion system: pCambia2300-GFP 2 μg, BamH Ⅰ 2 μg, EcoRI 2 μg, rCutSmart 5 μg and the remainder ddH2O; (2) Obtaining the target fragment: Primers (SEQ ID No. 3 and SEQ ID No. 4) were designed using the cotton genome sequence as a reference, and the target fragment was obtained by PCR amplification. GhbZIP208 Full-length gene. Using cDNA from 15-day-old cotton fiber (cotton fiber tissue 15 days after flowering) as a template, the gene with restriction sites was amplified using Novizan's high-fidelity enzyme 2×PhantaMax Master Mix (Dye Plus). GhbZIP208 Full-length gene.

[0050] SEQ ID No.3: acgggggactcttgaggatccATGGAAGTGCCTGGATTTGATG; SEQ ID No.4: ccgggtaccgagctcgaattcAGAAGCTGATTTAGGTTTCATGGAG; 50 μL PCR reaction system: 25 μL of 2×Phanta Max Master Mix (Dye Plus), 2 μL of template, 2 μL each of forward and reverse primers, and the remainder ddH2O.

[0051] PCR amplification program: 95 ℃ pre-denaturation for 3 min; 95 ℃ denaturation for 30 s, 58 ℃ annealing for 15 s, 72 ℃ extension for 30 s, 35 cycles; 72 ℃ extension for 5 min.

[0052] (3) Constructing a carrier The linearized vector and the target fragment were homologously recombined using the ClonExpress Ultra One Step Cloning Kit from Nanjing Novizan Biotechnology Co., Ltd. to construct the vector, which was then transformed into Escherichia coli and plated on LB medium containing 10 μg / mL Kan+ to obtain the pCambia2300-GhbZIP208 recombinant plasmid.

[0053] The StarPrep Fast Plasmid Mini Kit from Kangrun Company was used to extract plasmids from Escherichia coli. The recombinant plasmids were introduced into Agrobacterium tumefaciens GV3101 to obtain recombinant Agrobacterium. The recombinant Agrobacterium was plated on LB medium containing 10 μg / mL Kan+ and 5 μg / mL Rif+. Single clones were picked and positive strains were detected for use in cotton genetic transformation.

[0054] Example 6: Subcellular Gene Localization Tobacco was grown under 16 h / 8 h light / dark conditions, and a resuspension was prepared for infection when the tobacco reached the appropriate age. The OD of the bacterial culture was measured using the resuspension. 600 The pH was adjusted to 0.8-1.2, and the prepared solution was then allowed to stand in the dark for 3 hours. After 3 hours, the bacterial solution was injected into the underside of tobacco leaves, and the solution was kept in the dark for 24 hours. One day after the light was restored, the subcellular localization of the GhbZIP208 protein was observed using a laser confocal microscope.

[0055] Preparation of 100 mL resuspension: 1 mL 1 M MgCl2, 1 mL 1 M MES, 100 μL 200 mM acetylsyl syringone (AS) and the remainder ddH2O.

[0056] The results are as follows Figure 4As shown, the green fluorescence of the control group was uniformly distributed in the cell membrane, nucleus, and cytoplasm of tobacco cells, while the green fluorescence of the experimental group was distributed in the nucleus of tobacco leaf cells. The red fluorescence used for nuclear marker localization was localized only in the cell nucleus.

[0057] Example 7 Transformation of genetically modified cotton Seeds of material 75 were soaked in a clean bench, and the stem tips were separated. The stem tips were then soaked in a solution containing... GhbZIP208 In the Agrobacterium infection solution containing gene editing vectors and overexpression vectors, the cells were sonicated for 40 s, the bacterial solution was discarded and dried for 10 min. Subsequently, the cells were sequentially immersed in the bacterial solution at 37℃ for 40 s, immersed at 80 rpm for 50 min, and dried for 10 min. Then, the shoot tips were transferred to co-culture medium (30 g / L glucose + 3 g / L plant gel + 4.44 g / L M404, pH adjusted to 6.04-6.06, sterilized and cooled, and then 5 μL of 200 mM AS was added) and cultured in the dark for 3 days. After co-culture, the cells were transferred to induction medium (MS + 2 mg / L 6-BA + 0.1 mg / L NAA + 4 mg / L AgNO3 + 3% sucrose + 300 mg / L termethin). The medium was changed every 15 days until differentiation into seedlings was achieved.

[0058] Example 8: Cultivation and Identification of Gene-Edited Cotton Primers targeting the pKSE401 vector sequence were designed for the DNA of the obtained mutant T0 generation lines. Plants that showed a corresponding band after agarose gel electrophoresis were selected. Specific primers were then designed flanking the gRNA1 and gRNA2 mutation sites. Positive plants were verified in plants containing the pKSE401 vector. The amplified specific fragments were sequenced using the Hi-Tom method. Figure 5 The sequencing results were compared with the reference sequence, and those with site mutations near the sgRNA were considered positive. These positive T0 generation plants were then propagated in the field to obtain T4 generation positive plants for phenotypic identification.

[0059] cas9-pKSE401-F (SEQ ID No. 17): 5'-TGAGCTTGCCACGTGTGTTA-3'; cas9-pKSE401-R (SEQ ID No. 18): 5'-TTTGTTGGTCGCCGTTAGGA-3'; Hi-Tom-bZIP208-F (SEQ ID No. 19): 5'-ggagtgagtacggtgtgcCGACTATAAAACCTTGAATCCATCTT-3'; Hi-Tom-bZIP208-R (SEQ ID No. 20): 5'-gaggttggatgctggatggGAAAGCTGACGATTAATTGTTTGAA-3'.

[0060] Example 9: Cotton culture and identification with gene overexpression For the DNA of the T0 generation overexpression line, the upstream detection primer was designed in the 35S promoter region, and the downstream primer was the gene itself. The negative control showed no target band, while the positive control showed a clear band at the target location. Figure 6 These positive T0 generation plants were further propagated in the field to obtain T4 generation positive plants for phenotypic identification.

[0061] bZIP208-F (SEQ ID No. 21): 5'- GACGCACAATCCCACTATCC-3'; bZIP208-R (SEQ ID No. 22): 5'-CTGAGAGGTGCAAACACATCAG-3'.

[0062] Example 10: Phenotypic Identification of Transgenic Cotton Fibers To assess the expression effect of transgenic materials in fibers, mature cotton bolls from wild-type WT, mutant, and overexpression plants were harvested from the field, with three biological replicates from each line. Fiber length at maturity was photographed on T4 generation cotton plants. Figure 7 a) and statistical analysis ( Figure 7 (b) The results show that GhbZIP208 The mutant plants had longer fibers than the WT plants. Overexpression GhbZIP208 The fibers of the gene-expressing plants were shorter than those of the WT plants. There were significant differences among the overexpressing plants, wild-type plants, and mutants.

[0063] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A type of regulation GhbZIP208 Application of gene expression reagents in regulating cotton fiber quality and yield.

2. The application according to claim 1, characterized in that, The cotton fiber quality and yield include the length of the cotton fibers.

3. The application according to claim 2, characterized in that, The regulation includes downgrading or eliminating the... GhbZIP208 Genes that promote the elongation of cotton fibers.

4. The application according to claim 1 or 3, characterized in that, The GhbZIP208 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No.

1.

5. A method for increasing cotton fiber length, characterized in that, This includes downregulation or knockout in the target cotton genome. GhbZIP208 Gene.

6. The method according to claim 5, characterized in that, The GhbZIP208 The nucleotide sequence of the gene is shown in SEQ ID No.

2.

7. A method for cultivating a long-fiber cotton variety, characterized in that, This includes downregulation or knockout in the target cotton genome. GhbZIP208 Gene.

8. The cultivation method according to claim 7, characterized in that, The downsizing or knockout GhbZIP208 Genetic methods, including constructing GhbZIP208 The gene is linked to a gene-editing vector, and the recombinant gene vector is transformed into the target cotton genome using an Agrobacterium-mediated genetic transformation method.

9. A method for breeding long-fiber cotton varieties, characterized in that, In the cotton genome GhbZIP208 Gene expression levels were detected, and varieties with lower expression levels were cotton varieties with longer fibers.

10. The method according to claim 9, characterized in that, The detection GhbZIP208 The primer pair for gene knockout includes an upstream primer with nucleotide sequences as shown in SEQ ID No. 5 and a downstream primer as shown in SEQ ID No. 6.

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