Protein for improving cotton fiber length and use thereof

Abstract

The application discloses a protein for improving cotton fiber length and application thereof, and belongs to the field of botany. The protein comprises one or any combination of a GhARF2 protein with an amino acid sequence as shown in SEQ ID NO. 2, a GhGASA4 protein with an amino acid sequence as shown in SEQ ID NO. 4 and a GhGASA24 protein with an amino acid sequence as shown in SEQ ID NO. 6. The application studies the functions of GhARF2, GhGASA4 and GhGASA24 genes in cotton fiber development through molecular biology experiments, plant tissue culture technology and the like, proves that overexpression of the above genes can significantly promote fiber development and plays an important role in fiber development, provides a theoretical basis for cotton fiber development, effectively widens cotton breeding gene resources and provides high-quality germplasm resources for cotton breeding.

Description

Technical Field

[0001] This invention relates to the field of botany, and in particular to a protein for improving cotton fiber length and its applications. Background Technology

[0002] Cotton is an important economic crop, providing the market with a large amount of natural textile raw materials. As a major cotton producer and consumer, my country still relies heavily on imports for high-quality raw cotton. Therefore, improving cotton fiber quality remains a crucial issue in cotton breeding. Cotton fiber is an extremely elongated single cell formed from the differentiation and development of epidermal cells of the ovule. Cotton fiber development generally includes four distinct yet overlapping stages: fiber initiation, fiber elongation, secondary cell wall thickening, and maturity. The life activities at each stage of fiber development affect the yield and quality of cotton fiber, especially the fiber elongation and secondary cell wall thickening stages, which are most closely related to fiber development and quality formation. The specific stages of fiber development are determined by key genes, and the spatiotemporal specificity of gene expression is mainly achieved through the interaction between transcription factors and the promoters of downstream genes. Therefore, transcription factors play a vital role in fiber development.

[0003] ARF proteins are a class of key transcriptional regulators in plant development. The gene family contains multiple members and mainly controls various processes of plant growth and development. Their functions are primarily regulated by auxin IAA. Cotton fibers are a type of extremely elongated single-celled structure. Currently, there are no reports on the function of cotton GhARF2 protein in regulating cotton fiber elongation. Further research is needed in this field on the interaction between GhARF2 and downstream gene promoters to regulate the expression of downstream genes and further promote the biological function of fiber elongation. Summary of the Invention

[0004] The purpose of this invention is to provide a protein that improves cotton fiber length and its applications, thereby addressing the problems existing in the prior art. This invention discloses for the first time that overexpression of cotton genes GhARF2, GhGASA4, and GhGASA24 all promote cotton fiber elongation, providing a theoretical basis for cotton fiber development and effectively expanding cotton breeding gene resources.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] The present invention provides a protein for improving cotton fiber length, the protein comprising one or any combination of GhARF2 protein with amino acid sequence as shown in SEQ ID NO.2, GhGASA4 protein with amino acid sequence as shown in SEQ ID NO.4, and GhGASA24 protein with amino acid sequence as shown in SEQ ID NO.6.

[0007] Furthermore, the gene sequence encoding the GhARF2 protein is shown in SEQ ID NO.1; the gene sequence encoding the GhGASA4 protein is shown in SEQ ID NO.3; and the gene sequence encoding the GhGASA24 protein is shown in SEQ ID NO.5.

[0008] The present invention also provides a gene for improving cotton fiber length, the gene comprising one or any combination of the GhARF2 gene with nucleotide sequence as shown in SEQ ID NO.1, the GhGASA4 gene with nucleotide sequence as shown in SEQ ID NO.3, and the GhGASA24 gene with nucleotide sequence as shown in SEQ ID NO.5.

[0009] Furthermore, the gene also includes a gene that has more than 90% homology with the GhARF2, GhGASA4 and GhGASA24 genes and encodes GhARF2, GhGASA4 and GhGASA24.

[0010] The present invention also provides a biomaterial containing the gene for improving cotton fiber length, the biomaterial comprising a vector, cells, and transgenic cotton.

[0011] The present invention also provides a method for improving cotton fiber length by introducing a vector containing the aforementioned gene into cotton to upregulate the content of the protein.

[0012] Furthermore, the vector includes insertion sites for genes GhARF2, GhGASA4, and GhGASA24.

[0013] The present invention also provides the use of the protein or gene described herein in the preparation of reagents or combinations that promote the elongation of cotton fibers.

[0014] The present invention also provides the application of the protein or gene described herein in regulating cotton fiber development.

[0015] The present invention also provides the application of the described protein or the described gene in the cultivation of transgenic cotton.

[0016] Furthermore, the cultivation of transgenic cotton includes introducing a vector containing the gene into cotton, inducing and screening callus tissue with upregulated protein content, and cultivating transgenic cotton that promotes cotton fiber development through plant tissue culture technology.

[0017] The present invention discloses the following technical effects:

[0018] This invention discloses for the first time that overexpression of cotton genes GhARF2, GhGASA4, and GhGASA24 all promote cotton fiber elongation; a comparison of cotton bolls from GhARF2, GhGASA4, and GhGASA24 overexpressing plants with wild-type J668 showed that the mature fibers of the overexpressing plants were significantly larger than those of the wild-type plants.

[0019] This invention discloses for the first time that the cotton gene GhARF2 promotes the expression of downstream genes GhGASA4 and GhGASA24 by binding to the promoters of GhGASA4 and GhGASA24 respectively, thereby further promoting cotton fiber development. This was verified by biological experiments such as Chip-Seq, yeast single-hybrid and dual-luciferase assays.

[0020] This invention demonstrates that genes such as GhARF2, GhGASA4, and GhGASA24 regulate cotton fiber length and play a crucial role in fiber development; overexpression of GhARF2, GhGASA4, and GhGASA24 significantly promotes fiber development. Their functions in cotton fiber development were investigated through molecular biology experiments and plant tissue culture techniques. This provides a theoretical basis for cotton fiber development, effectively expands cotton breeding gene resources, and provides high-quality germplasm resources for cotton breeding. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 To determine the expression level of the GhARF2 gene in cotton tissues at different stages, the transgenic local material J668 was used. Ovules with 3 and 5 DPA and fibers with 10, 15, 20, and 25 DPA were taken for quantitative analysis.

[0023] Figure 2 This section presents statistical data on fiber phenotypes, fiber length, and ARF2 expression levels in different transgenic cottons. A represents the quantitative detection of ARF2, CK represents 15DPA fibers from the recipient material J668, and ARF2-OE represents 15DPA fibers overexpressing ARF2, each representing three biological replicates. B and C represent the phenotypes and fiber length statistics (bar = 1 mm) of mature fibers from the control (transgenic recipient J668, CK), GhARF2 gene overexpression (ARF2-OE), and GhARF2 gene knockout (ARF2-Cas9), respectively, each representing three biological replicates.

[0024] Figure 3 This document presents the ChIP-Seq results and enrichment typing of transgenic GhARF2. A represents the ChIP-Seq results of cotton fibers overexpressing ARF2, showing sequences in the genome that can bind to the transcription factor ARF2, including gene promoter regions. B represents the gene corresponding to the transcription start site closest to the midpoint of the peak, used for gene ontology (GO) analysis.

[0025] Figure 4 In Chip-Seq experiments for GhARF2 gene overexpression, it can bind to the upstream promoters of GhGASA4 and GhGASA24.

[0026] Figure 5 The following assays were performed to verify the interaction between GhARF2 and the upstream 2K promoters of GhGASA4 and GhGASA24 using yeast single-hybrid and dual-luciferase assays. Specifically: A) Verification of the interaction between GhARF2 and the upstream 2K promoters of GhGASA4 and GhGASA24 using yeast single-hybrid assays; B) Verification of the interaction between GhARF2 and the upstream 2K promoter of GhGASA4 using dual-luciferase assays; C) Verification of the interaction between GhARF2 and the upstream 2K promoter of GhGASA24 using dual-luciferase assays; DG) Detection of which segment of the upstream 2K promoter of GhGASA4 interacts with GhARF2 by dividing the GhGASA4 upstream 2K promoter into 5 segments and verifying which segment of GhGASA24 interacts with GhARF2 using dual-luciferase assays; HK) Detection of which segment of the upstream 2K promoter of GhGASA24 interacts with GhARF2 by dividing the GhGASA24 upstream 2K promoter into 5 segments and verifying which segment of GhGASA24 interacts with GhARF2 using dual-luciferase assays.

[0027] Figure 6 Statistical data on fiber phenotypes, fiber length, and expression levels of GhGASA4 and GhGASA24 in different transgenic cottons are presented. Specifically, AC represents the fiber expression levels, fiber length phenotypes, and fiber length statistics (bar = 1 mm) of cotton at the critical stage 15DPA in the control (transgenic recipient J668, WT), GhGASA4 gene overexpression (GASA4 OE), and GhGASA4 gene interference (GASA4 RNAi) groups, respectively. DF represents the fiber expression levels, fiber length phenotypes, and fiber length statistics (bar = 1 mm) of cotton at the critical stage 15DPA in the control (transgenic recipient J668, WT), GhGASA24 gene overexpression (GASA24 OE), and GhGASA24 gene interference (GASA24 RNAi) groups, respectively.

[0028] Figure 7This study investigated the expression levels of key cellulose-related genes, cell wall thickness, and cellulose content in different GhGASA24 transgenic cottons. Specifically, A and B represent the expression changes of two key cellulose synthase genes in fibers (5 DPA, 10 DPA, 15 DPA, and 20 DPA) at different growth stages in cotton from the control (transgenic recipient J668, WT), GhGASA24 gene overexpression (GASA24 OE1-3), and GhGASA24 gene interference (GASA24 RNAi1-3), respectively. CG represents the expression levels of key cellulose-related genes in the control (transgenic recipient J668, WT), GhGASA24 gene overexpression (GASA24 OE1-3), and GhGASA24 gene interference (GASA24 RNAi1-3). Comparison of cell wall thickness and related statistics (bar = 10 μm) of cotton at different growth stages (5, 10, 20, 30 DPA) using RNAi1-3; H represents the control (transgenic receptor J668, WT), and the comparison of cellulose content at different growth stages (5, 10, 20, 30 DPA) of cotton with GhGASA24 gene overexpression (GASA24OE1-3) and GhGASA24 gene interference (GASA24RNAi1-3). Detailed Implementation

[0029] Through extensive and in-depth research, the inventors have for the first time discovered a GhARF2 transcription factor and its downstream genes GhGASA4 and GhGASA24 that can regulate the length of cotton fibers / plant cells. Experiments show that overexpression of GhARF2 in cotton promotes fiber cell elongation, while knockout of GhARF2 inhibits fiber cell elongation, indicating that GhARF2 plays a crucial positive regulatory role in cotton fiber elongation. This invention discloses the function and uses of the cotton GhARF2 transcription factor protein, particularly its positive effects on promoting fiber cell elongation and improving cotton fiber length and quality traits, demonstrating broad application prospects.

[0030] In their research on cotton fiber development, the inventors cloned a gene, GhARF2 (Gh_D11G042800), which encodes an ARF-like protein and is specifically highly expressed during the fiber cell elongation phase. To further investigate the biological function of GhARF2, the inventors conducted transgenic cotton functional analysis, constructed vectors for GhARF2 overexpression and Cas9 expression, transformed cotton, and successfully obtained multiple transgenic lines for each vector. Analysis of transgenic plants grown in greenhouses and fields revealed that overexpression of GhARF2 in cotton promotes fiber cell elongation, while knockout of GhARF2 expression inhibits fiber cell elongation, indicating that GhARF2 plays a crucial positive regulatory role in cotton fiber elongation. Gene expression and various molecular biological verifications showed that GhARF2 regulates the expression of downstream genes GhGASA4 (Gh_A06G023500) and GhGASA24 (Gh_D04G182700). Simultaneously, overexpression and RNA interference (RNAi) of GhGASA4 and GhGASA24 revealed that both GhGASA4 and GhGASA24 play positive regulatory roles in cotton fiber elongation. These results indicate that transcription factors GhARF2 and their downstream genes GhGASA4 and GhGASA24 are important regulators of cotton fiber cell development, possessing significant potential and application value in promoting cotton fiber elongation and improving fiber quality.

[0031] Regarding the cotton GhARF2 gene, the inventors conducted the following research: through transgenic genetic transformation and protein-protein interaction experiments, they elucidated the mechanism by which GhARF2 regulates cotton fiber cell development. This invention discloses for the first time the information and applications of GhARF2 and its downstream genes GhGASA4 and GhGASA24. Surprisingly, they discovered that regulating its expression level can regulate cotton fiber cell growth; in particular, overexpression of GhARF2 and its downstream genes GhGASA4 and GhGASA24 promotes fiber cell growth and increases cotton fiber length, which has significant application value for improving cotton fiber quality.

[0032] The GhARF2, GhGASA4, and GhGASA24 genes of this invention can be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The genomic DNA can be identical to the sequence shown in SEQ ID NO.1 or a degenerate variant.

[0033] The carrier used in the embodiments of this invention:

[0034] pCAMBIA2300GFP vector was purchased from Shanghai Qiming Biotechnology Co., Ltd.; pRGEB32 vector was purchased from Addgene; pHIS2 vector was purchased from Puruting Biotechnology (Beijing) Co., Ltd.; pSK vector was purchased from BioWind Co., Ltd.; pBI121 vector was purchased from Shanghai Maokang Biotechnology Co., Ltd.

[0035] pGhGASA4-pHIS2 and pGhGASA24-pHIS2 were achieved by inserting the pGhGASA4 promoter (SEQ ID NO.23) and pGhGASA24 promoter (SEQ ID NO.24) into the Eco1 site of the pHIS2 vector via single enzyme digestion; GhARF2-pGADT7-Rec2 was achieved by inserting the GhARF2 (SEQ ID NO.1) sequence into the Eco1 site of the pGADT7-Rec2 vector via single enzyme digestion; 0800-ARF2 was achieved by inserting the GhARF2 (SEQ ID NO.1) gene into the pGreenII 0800 vector via homologous recombination after double digestion with Kpn1 and Pst1; pGASA4-62sk and pGASA24-62sk were achieved by double digestion with Xba1 and Spe1 into the pGreenII 62-SK vector via homologous recombination to insert the pGhGASA4 promoter and pGhGASA24 promoter.

[0036] Example 1: Isolation and Identification of the GhARF2 Gene (cDNA) Sequence

[0037] 1. Quantitative RT-PCR analysis of GhARF2 gene expression

[0038] Germplasm resources: Upland cotton J668 (provided by the cotton germplasm resource mid-term bank of the Cotton Research Institute of the Chinese Academy of Agricultural Sciences, J668 material).

[0039] Tissue samples: J668 cotton ovules 3 days after flowering (3PDA ovules), ovules 5 days after flowering (5PDA ovules), fibers 10 days after flowering (10PDA fibers), fibers 15 days after flowering (15PDA fibers), fibers 20 days after flowering (20PDA fibers), and fibers 25 days after flowering (25PDA fibers).

[0040] Total RNA was extracted from cotton tissue samples (according to Chen BJ, 2021. GhGASA10-1 promotes the cellelongation in fiber development through the phytohormones IAA-induced, BMCPlant Biology, 2021, 21(1): 1-15).

[0041] 2. Real-time quantitative RT-PCR for gene expression studies

[0042] The specific method is based on He SP, 2021. The genomic basis of geographic differentiation and fiber improvement in cultivated cotton, Nature Genetics, 2021, 53(6):916-924.

[0043] Total RNA extracted in step 1 (2 μg / sample) was reverse transcribed into cDNA using M-MLVRNase HReverse Transcriptase (Promega). Then, using the cDNA as a template, quantitative PCR was performed with gene-specific primers (quantitative primers included GhARF2 / GhGASA4 / GhGASA24 / GhCESA10-1 / GhCESA4-4, and internal control primers) and Real-time PCR Master Mix (TOYOBO, Japan). The cotton GhUBQ gene was used as an internal standard for the RT-PCR reaction. The reaction program was: 95°C for 30 s, denaturation at 95°C for 10 s, annealing at 60°C for 30 s, for 40 cycles. The reaction mixture consisted of: 10 μl MIX, 7 μl ddH2O, 0.5 μl F / R primers, and 2 μl cDNA. Amplification of the target gene in each cycle was detected by SYBR-Green fluorescence. Specific primer information is shown in Table 1.

[0044] Table 1. Specific sequences of each primer

[0045]

[0046]

[0047] As an upstream transcription factor of GhGASA4 and GhGASA24, the GhARF2 gene was used to illustrate fiber expression levels at different stages. Real-time quantitative RT-PCR analysis revealed that GhARF2 gene expression was upregulated during the secondary wall thickening stage of cotton fibers (approximately 20 days after flowering). Figure 1Therefore, GhARF2 was cloned from cotton, and other downstream genes showed high expression levels in fibroblast thickening. The CDS sequence of GhARF2 was obtained through DNA and protein sequence analysis, as shown in SEQ ID NO. 1, and the amino acid sequence of the protein encoded by this gene is shown in SEQ ID NO. 2. The CDS sequences of the downstream functional genes GhGASA4 and GhGASA24 are shown in SEQ ID NO. 3 and 5, respectively, and the amino acid sequences of the proteins encoded are shown in SEQ ID NO. 4 and 6, respectively.

[0048] SEQ ID NO.1GhARF2 CDS sequence:

[0049] ATGACTACGTCGGAGATATCGATAAAAGGAAATTGTGTCAACGGAAGAGGAGATAGTTTTTCTTCCGGTTATACCGAGCCACGAGATACTAGGAACGCCATGGAAGGGCAGAACGGTCATTCCGCTCGTACAGCTGCCGTCAGAGAAACCGTAGACCCCGAAAGGGCGCTGTATACG GAGCTATGGCATGCATGTGCTGGACCTCTGGTGACGGTCCCTCGCGAATTAGAGCGCGTGTTCTACTTTCCTCAAGGTCACATAGAACAGGTTGAGGCGTCTACTCATCAGGTATCAGACCAGCAGATGCCGGTGTATGACCTTCCACCAAAGATCCTTTGTCGTGTGATTAACGTA CAACTAAAGGCTGAACTGGATACTGATGAGGTTTTTGCTCAAGTGACTTTGCTTCCTGAACATAATCAAGATGAGAACATGGTGGACAAGGAGCCTCCCATTCTTGAACCCCCTCGGTTCCAAGTGCATTCGTTTTGCAAAACCCTGACTGCTTCAGATACGAGTACCCATGGTGGA TTTTCAGTGCTCAGGCGGCATGCCGATGAATGTCTTCCACCACTGGATATGTCGCTGCAACCTCCAACACAGGAGCTGGTTTCTAAGGATTTGCATGGAAATGAGTGGCGATTCCGGCATATCTTCAGGGGGTCAGCCACGAAGACACTTGCTTCAAAGCGGTTGGAGTGTTTTTGTTA

[0050] GCTCCAAGAAGCTTGTTGCTGGGGATGCATTTATATTTTTAAGAGGCGAGAATGGAGAAT

[0051] TATGCGTTGGTGTACGGCGAGCATTGAGACAACAGGGCAATGTTCCTTCATCGGTTATAT

[0052] CAAGTCATAGCATGCATCTTGGTGTGCTAGCGACAGCATGGCATGCCTACACTACCAGAA

[0053] CCATATTCACTGTGTATTACAAACCCAGGACAAGTCCAGCTGAGTTCATTGTTCCATTTAA

[0054] TCAGTACATGGAGTCGGTAAAGAACAATTACTCAATAGGGATGAGGTTCAAAATGAGAT

[0055] TTGAAGGTGAAGAAGCTCCTGAACAGAGGTTTACTGGAACAATAGTTGGAATCGAAGA

[0056] TGCTGATCCAAAAAGGTGGCAGGGTTCCAAATGGAGATGCCTGAAGGTGCGATGGGAT

[0057] GAAACGTCTACAATACCTCGTCCAGAGAGAGTTTCTCCTTGGAAAATTGAACATGCTTT

[0058] GTCTCCTCCTGCCCTTAATCCCCTTCCAATGCCCCGGCCAAAAAGGCCTCGAACTAATGC

[0059] TGTATCTTCATCCCCTGATTCCTCTGTACTTAGTAGGGAAGGTTCTTCCAAAGTTACTGTA

[0060] GACCCTTTGCCGGCCAGTTCATTTTCAAGGGTCTTGCAAGGTCAAGAATTCTCGACCTT

[0061] GAGAGGCACATTTGCTGAGAGTAATGATTCTGAAACTGCTGATAGGTCAGTGATGTGGC

[0062] CACCTTCAATAGATGATGAGAAGATTGATGTAGCTCATGGTGAAAGAAAATTTGGGTCA

[0063] GAGAATTGGATGCCCTCTAGGAGGCATGAACCAACTTACACAGATTTGCTCTCAGGTTTT

[0064] GGGTCGAATGCTGATACATCGCGCGGATATTATCCTTCCTTTGTTGATCAAACTTCAGTAG

[0065] CTGGTAATTCGGGGAAAAAACAATTACTAGGTCAAGAAGGGAAGCTTGGCTCTTGGTCC

[0066] CTCCTGCCATCTGGTCTCTCACTCAAGTTGTCTGACAGTAGTACAGACCCTCCTTTGCAA

[0067] GGTTCTGATGTGCCTTGTCAGGCGCGGGGAAATGGTAGATTTAGTGGTTTTGGTGACTAC

[0068] CCTATACTTGAAGGTCGTAGGATTGAATGCTCACGTGGTAATTGGTTGATGCCTCCCCCA

[0069] ACCACTTCTTGTTATGATAATTCAATCCAGTCAAGAGATTTAATGCCGAAAACATCATTGG

[0070] CTCAAGAGCATAAGAATGGAAAATCTAGAGAAGGAAACTGCAAGCTCTTTGGTATTCCT

[0071] CTCATAAGTGCTTCTAGCGCATCAGAGCCTGCAGTCTCTCATATTAGTGCTTTCGCCAAG

[0072] CCTGTAGGACATATGCAAGCTGCATTGCACCAGGTTCATGCACTTGAATCTGATAAAAGG

[0073] TCTGAAAATTCAAACGCCTCCCAGATGGCAGAGGATGTTTCTGCTTTTAATGAGCAGGA

[0074] GAAAATAGTGAAGCTGGGTCAGCCCCATGCACGGGAGTTTCAAAGCAAACTGTCTACT

[0075] GCTTCAACTAGGAGTTGTACTAAGGTTCTCATGCAGGGGACTGCTCTTGGAAGGTCTGT

[0076] GGACCTTACCAAGTTCAACAACTATGATGAGTTGATCGCTGAATTGGATCAATTATTTGA

[0077] GTTTGGAGGTGAATTAATGGCCCCTCAAAAGAACTGGCTTGTTGTTTATACTGATGATGA

[0078] GGGTGATATGATGCTTGTTGGCGATGATCCTTGGCAGGAATTTTGTGCCATGGTCCGCAA

[0079] GATTGGTATCTACACTAGGGAAGAGGTCCAGAAGATGAAGCCAGGGTCGTTGGGTTCAA

[0080] AGTTTGAGGACATTCCAGTTCCCACAGAAGGTACAGTTGCAAAAGAAGTGAACTGTCC ATCAGCATCTAGTGCAAAGAATTGTTCAGGGTAA。

[0081] SEQ ID NO.2 GhARF2 protein sequence:

[0082] MTTSEISIKGNCVNGRGDSFSSGYTEPRDTRNAMEGQNGHSARTAAVRETVDPERALYTELWHACAGPLVTVPRELERVFYFPQGHIEQVEASTHQVSDQQMPVYDLPPKILCRVINVQLKAELDTDEVFAQVTLLPEHNQDENMVDKEPPILEPPRFQVHSFCKTLTASDTSTHGGFSVLRRHADECLPPLDMSLQPPTQELVSKDLHGNEWRFRHIFRGQPRRHLLQSGWSVFVSSKKLVAGDAFIFLRGENGELCVGVRRALRQQGNVPSSVISSHSMHLGVLATAWHAYTTRTIFTVYYKPRTSPAEFIVPFNQYMESVKNNYSIGMRFKMRFEGEEAPEQRFTGTIVGIEDADPKRWQGSKWRCLKVRWDETSTIPRPERVSPWKIEHALSPPALNPLPMPRPKRPRTNAVSSSPDSSVLSREGSSKVTVDPLPASSFSRVLQGQEFSTLRGTFAESNDSETADRSVMWPPSIDDEKIDVAHGERKFGSENWMPSRRHEPTYTDLLSGFGSNADTSRGYYPSFVDQTSVAGNSGKKQLLGQEGKLGSWSLLPSGLSLKLSDSSTDPPLQGSDVPCQARGNGRFSGFGDYPILEGRRIECSRGNWLMPPPTTSCYDNSIQSRDLMPKTSLAQEHKNGKSREGNCKLFGIPLISASSASEPAVSHISAFAKPVGHMQAALHQVHALESDKRSENSNASQMAEDVSAFNEQEKIVKLGQPHAREFQSKLSTASTRSCTKVLMQGTALGRSVDLTKFNNYDELIAELDQLFEFGGELMAPQKNWLVVYTDDEGDMMLVGDDPWQEFCAMVRKIGIYTREEVQKMKPGSLGSKFEDIPVPTEGTVAKEVNCPSASSAKNCSG。

[0083] SEQ ID NO.3 GhGASA4 CDS sequence:

[0084] ATGAAGATGGTATTGGTGCTTTTCTTGCTTGTTTCTCTTGCTCTCAGCTCTTGTTTCTTCGAGGTGTCGATTGCCGGTTCGGATTTTTGTGACTCAAAGTGTGCGGTGAGGTGCTCAAAGGCAGGGGTTCAAGACAGGTGTTTGAAATATTGTGGGATTTGTTGTGAGAAATGTCATTGTGTTCCATCTGGGACATTTGGGCATAAAGATGAATGCCCTTGTTATAGGGACATGAAGAACTCTAAGGGCAAATCCAAGTGCCCTTAG。

[0085] SEQ ID NO.4 GhGASA4 protein sequence:

[0086] MKMVLVLFLLVSLALSSCFFEVSIAGSDFCDSKCAVRCSKAGVQDRCLKYCGICCEKCHCVPSGTFGHKD ECPCYRDMKN SKGKSKCP。

[0087] SEQ ID NO.5 GhGASA24 CDS sequence:

[0088] ATGAAGCTCTTGTTTCTAACTTTGCTGCTTTGTTCTCTTCTTCTATGTTCTTCAGTTTTTGCACCAACAATGGCTCAGCCTCGTTCACCTTTTTGTGAAGGGAAATGCAAAGGGAGGTGCAATAAAGCGGCGGTTTGGGATCGGTGCTTCAAATATTGCGGCATATGTTGCGAGGAGTGTCAATGCGTTCCGTCCGGTACTTACGGGAACAAACACGAGTGTCCTTGCTACAGAGAT AAGGTGAACAACAAGGGCAAACCCAAATGCCCTTGA。

[0089] SEQ ID NO.6GhGASA24 protein sequence:

[0090] MKLLFLTLLLCSLLLCSSVFAPTMAQPRSPFCEGKCKGRCNKAAVWDRCFKYCGICCE ECQCVPSGTYGNKHECPCYRDKVNNKGKPKCP。

[0091] Example 2: Construction of GhARF2 transgenic cotton and statistical analysis of fiber length

[0092] 1. Constructing plant expression vectors

[0093] The overexpression vector p2300-GhARF2 of the GhARF2 gene and the knockout vector pRGEB32-GhU6.7-NPT2-GhARF2 of the GhARF2 gene were constructed respectively.

[0094] The overexpression vector p2300-GhARF2 was obtained by double digestion of the pCAMBIA2300GFP vector at the BamHI and SacI sites. After the GFP tag was removed, the sequence shown in SEQ ID NO.1 was ligated into the vector using homologous recombination.

[0095] The vector pRGEB32-GhU6.7-NPT2-GhARF2, which knocks out the GhARF2 gene, is created using CRISPR / Cas9 gene editing technology. Homologous recombination is used to link a dual-target tRNA-PAM1-gRNA-tRNA-PAM2-gRNA sequence (SEQ ID NO. 22) to the pRGEB32 vector driven by ubiquitin U6.7, thereby knocking out the sequence shown in SEQ ID NO. 7.

[0096] For specific construction methods, refer to Tian ZL, 2022. Strigolactones act downstream of gibberellins to regulate fiber cell elongation and cell wall thickness incotton (Gossypium hirsutum).

[0097] SEQ ID NO.7GhARF2 knock out sequence:

[0098] GAACCCCCTCGGTTCCAAGTGCATTCGTTTTGCAAAACCCTGACTGCTTCAGATACGAGTACCCATGGTGGATTTTCAGTGCTCAGGCGGCATGCCGATGAATGTCTTCCACCACTGGATATGTCGCTGCAACCTCCAACACAGGAGCTGGTTTCTAAGGATTTGCATGGAAATGAGTGGCGATTCCGGCATATCTTCAGGGGTCAGCCACGAAGACACTTGCTTCAAAGCGGTTGGAGTGTTTTTGTTAGCTCCAAGAAGCTTGTTGCTGGGGATGCATTTATATTTTTAAGAGGCGAGAATGGAGAATTATGCGTTGGTGTACGGCGAG。

[0099] SEQ ID NO.22:

[0100] AACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCACCCCTCGGTTCCAAGTGCATTCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCAGAGAATTATGCGTTGGTGTACGGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC。

[0101] 2. Construction of transgenic plants

[0102] The constructed p2300-GhARF2 and pRGEB32-GhU6.7-NPT2-GhARF2 vectors were transformed into *Agrobacterium tumefaciens* LBA4404 via electroporation. Using upland cotton J668 as the recipient material, *Agrobacterium tumefaciens* was used to infect cotton hypocotyl explants. After co-culturing for 2 days, the hypocotyls were transferred to a selective medium for induction and screening of transformed callus tissue. After 8-10 months of subculture, the screened callus tissue was induced to differentiate into embryogenic callus tissue and somatic embryos. The somatic embryos then germinated and regenerated into transgenic cotton seedlings. The cotton seedlings were transplanted into soil and allowed to grow and develop until flowering and fruiting. Genomic DNA was extracted from cotton plants, and transgenic cotton plants were identified by PCR using agarose gel electrophoresis (knockout transgenic plants do not contain the GhARF2 gene and therefore do not have the corresponding electrophoretic band; overexpressed transgenic plants were examined using the cotton 35S strong promoter primer (GACGCACAATCCCACTATCC) plus the gene's own primer GhARF2-R).

[0103] 3. Statistics on the fiber length of transgenic cotton plants

[0104] Five or more T0 generation cotton lines were obtained through overexpression and knockout of the GhARF2 gene. The fiber length of T3 generation cotton plants at maturity was photographed and statistically analyzed using seeds from two generations. Figure 2 The expression levels of GhARF2 in fibers during key periods (B and C) and C) Figure 2 (A). The results showed that high expression of GhARF2 promoted fiber length elongation.

[0105] Example 3: Chip-Seq sequencing of GhARF2 transgenic cotton fibers

[0106] Chip-Seq sequencing steps:

[0107] (1) Formaldehyde cross-links the entire cell line (fiber), that is, it links the target protein to the chromatin;

[0108] (2) Separate the genomic DNA and break it into small fragments of a certain length using ultrasound;

[0109] (3) Add an antibody specific to the target protein, which forms an immunoprecipitation-immunobinding complex with the target protein;

[0110] (4) Remove cross-links and purify DNA to obtain a DNA sample with chromatin immunoprecipitation, ready for sequencing;

[0111] (5) Perform deep sequencing on the prepared samples.

[0112] To examine which downstream genes are directly regulated by GhARF2, chromatin immunoprecipitation sequencing (ChIP-Seq) was performed. We used Flag antibody and protein G MagBeads to immunoprecipitate (IP) the protein-DNA complex in 15 DPA transgenic fibers and degrade the ARF2 protein. We sequenced two IPs and one control library. In the IP experiments, there were 2509 peaks on the chromosomes (…). Figure 3 (A). To further explore the binding site characteristics of protein modifications and understand the regulatory mechanisms of protein modifications on genes, we identified the genes corresponding to the transcription start sites closest to the midpoint of the peak for gene ontology (GO) analysis. Figure 3 (B). These genes are involved in cell wall formation, plant hormone responses, and energy production, and are associated with fiber development. We also calculated the number of functional elements for each gene distributed across the genome. Approximately 7.69% of the peaks were located at gene promoters. Transcriptional profiling analysis showed that 28 candidate genes were strongly expressed during cotton fiber elongation and secondary cell wall thickness. Analysis of the promoters and genes above revealed peaks at the promoter sites of the GhGASA4 and GhGASA24 genes. Figure 4 The study also found that GhARF2 regulates cotton fiber elongation by binding to the promoters of these two genes and specifically expressing them in fibers.

[0113] Example 4: Yeast mono-hybrid and LUC experiments

[0114] First, the promoters pGhGASA4-pHIS2 and pGhGASA24-pHIS2 (2000 bp) and the gene GhARF2-pGADT7-Rec2 were constructed. These were co-transfected into yeast Y187, and the optimal concentration of 150 mM was selected using 3-AT screening. Then, the interactions between pGhGASA4-pHIS2 and GhARF2-pGADT7-Rec2, and between pGhGASA24-pHIS2 and GhARF2-pGADT7-Rec2, were verified in yeast. Figure 5 The vectors 0800-ARF2, pGASA4-62sk, and pGASA24-62sk were then constructed and transformed into Agrobacterium LBA4404, respectively. These vectors were then co-injected into tobacco. 0800-ARF2 and pGASA4-62sk showed fluorescence (A). Figure 5 B), 0800-ARF2 and pGASA24-62sk showed fluorescence ( Figure 5To further verify which binding element in pGhGASA4 and pGhGASA24 interacts with transcription factor ARF2, the promoters of pGhGASA4 and pGhGASA24 were segmented into 5 segments, and pHIS2 and 62sk vectors were constructed respectively to further verify which part of the promoter interacts with the transcription factor. Both methods were used to verify pGASA4P4 (C). Figure 5 DG) and pGASA24P3 Figure 5 The HK) interacts with the transcription factor ARF2. Analysis revealed that the binding element TGTCTC is present on the promoters of pGASA4P4 and pGASA24P3, indicating that ARF2 further regulates the expression of GhGASA4 and GhGASA24 by binding to the binding element TGTCTC in pGhGASA4 and pGhGASA24, thereby further regulating cotton fiber length.

[0115] Example 5: Construction of GhGASA4 and GhGASA24 transgenic cotton and statistical analysis of fiber length.

[0116] Plant expression vectors were constructed containing the overexpression vectors p2300-GhGASA4 and p2300-GhGASA24 of GhGASA4 (SEQ ID NO.3) and GhGASA24 (SEQ ID NO.5), as well as the GhGASA4-RNAi sequence (SEQ ID NO.8) and the GhGASA24-RNAi sequence (SEQ ID NO.9).

[0117] Construction methods of p2300-GhGASA4 and p2300-GhGASA24 vectors:

[0118] The pCAMBIA2300GFP vector was double-digested at BamHI and SacI sites to remove the GFP tag. Then, the coding sequences of GhGASA4 and GhGASA24 (shown in SEQ ID NO.3 and SEQ ID NO.5) were ligated into the vector using homologous recombination.

[0119] Methods for constructing vectors containing GhGASA4-RNAi and GhGASA24-RNAi sequences:

[0120] For the RNAi system, the first intron of GhTUB1 (SEQ ID NO. 25) was first amplified by PCR and inserted into the pSK vector to form a new pSK-TUAint vector. A specific sequence of approximately 300 bp from GhGASA4 and GhGSAS24 (shown in SEQ ID NO. 8-9) was ligated before the TUB1 intron, and the corresponding antisense strand was ligated after the TUB1 intron, forming a coding-intron-antisense recombination sequence. This sequence was then ligated between the BamH1 and Sac I sites of the pBI121 vector using homologous recombination, replacing the GUS tag of the pBI121 vector.

[0121] SEQ ID NO.8GhGASA4 RNAi sequence:

[0122] TGGTGCTTTTCTTGCTTGTTTCTCTTGCTCTCAGCTCTTGTTTCTTCGAGGTGTCGAT TGCCGGTTCGGATTTTTGTGACTCAAAGTGTGCGGTGAGGTGCTCAAAGGCAGGGGTTCAAGACAGGTGTTTGAAATATTGTGGGAT.

[0123] SEQ ID NO.9GhGASA24 RNAi sequence:

[0124] GCTCTTGTTTCTAACTTTGCTGCTTTGTTCTTCTTCTATGTTCTTCAGTTTTTCAC CAACAATGGCTCAGCCTCGTTCACCTTTTTGTGAAGGGAAATGCAAAGGGAGGTGCAAT AAAGCGGCGGTTTGGGATCGGTGCTTCAAATATTGCGGCATATGTTGCGAG.

[0125] SEQ ID NO.25:

[0126] GTAATTTTGGTTAATTTAAAGGTTCCATTTGAAGAGTTAAGTCCGGTTTATCTTTGAATTGAGCCTCTGTTTGGTTACAG.

[0127] The constructed vector was transferred into *Agrobacterium tumefaciens* LBA4404 via electroporation, followed by infection of cotton hypocotyls to obtain transgenic cotton (method as above). The transgenic cotton was identified by DNA extraction and PCR amplification. The fiber length at maturity and the concentration of GhGASA4 in the fibers at the critical 15-day post-conversion period (15 DPA) of the T3 generation cotton were statistically analyzed. Figure 6 A) and GhGASA24 ( Figure 6 The expression level of GhGASA4 (D) was determined by photographing mature fibers and statistical analysis. Figure 6 (B and C) and GhGASA24 ( Figure 6 The E and F components of GhGASA24 promote fiber elongation, respectively. Studies have found that GhGASA24 may promote fibroblast cell wall synthesis, and qPCR quantification results show that GhGASA24 promotes cellulose synthase GhCesA10-1 (…). Figure 7 A) and GhCesA4-4 ( Figure 7 The expression of B) was analyzed by paraffin sections of fibers at different time points (5 DPA, 10 DPA, 20 DPA, and 30 DPA) (experimental method as follows 1) and cellulose detection (method as follows 2). Figure 7 (CH) found that in 20DPA and 30DPA, GhGASA24 promoted cell wall thickening and increased cellulose synthesis.

[0128] 1. Paraffin Sections: Cotton fibers with 5, 10, 20, and 30 DPA were fixed with FAA solution and dehydrated in an ethanol series. The fiber ends closest to the ovules were embedded in paraffin wax and cut into 10 μm thick sections using a rotary microtome (Reichert-Histo stat, Germany) according to a published method (Huang et al., 2021). The sections were observed under a confocal laser scanning microscope (LSM710, Zeiss, Germany).

[0129] 2. Cellulose extraction and measurement were performed using a cellulose assay kit (Solarbio, China). The cellulose sample (0.1 g) was frozen and lyophilized in liquid nitrogen, then washed three times with ice-cold potassium phosphate buffer (0.5 mL, pH 7.0). The particles were washed with deionized water, dispersed in 80% ethanol, and incubated at 90°C for 20 min. After cooling to room temperature, they were centrifuged at 6000 g for 10 min. The particles were then washed twice, first with 80% ethanol and then with acetone. An appropriate amount of amylase was added to remove starch, the sample was centrifuged at 6000 g for 20 min, and the particles were dried at 50°C. The pellets were dissolved in H2O and H2SO4, respectively, and centrifuged for 10 min. The supernatant was then diluted with H2O, and working solution was added. The cellulose content was calculated by measuring the absorbance at 620 nm.

[0130] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for improving cotton fiber length, characterized in that, A vector containing a gene that improves cotton fiber length was introduced into cotton, thereby upregulating the content of the protein. The genes for improving cotton fiber length include one or any combination of the following: the GhARF2 gene with the nucleotide sequence shown in SEQ ID NO.1, the GhGASA4 gene with the nucleotide sequence shown in SEQ ID NO.3, and the GhGASA24 gene with the nucleotide sequence shown in SEQ ID NO.

5.

2. The method according to claim 1, characterized in that, The vector includes insertion sites for genes GhARF2, GhGASA4, and GhGASA24.

3. The application of a protein or its encoding gene that improves cotton fiber length in the preparation of reagents or combinations that promote cotton fiber elongation, characterized in that, The protein includes one or any combination of the following: GhARF2 protein with the amino acid sequence shown in SEQ ID NO.2, GhGASA4 protein with the amino acid sequence shown in SEQ ID NO.4, and GhGASA24 protein with the amino acid sequence shown in SEQ ID NO.6; The gene sequence encoding the GhARF2 protein is shown in SEQ ID NO.1; the gene sequence encoding the GhGASA4 protein is shown in SEQ ID NO.3; and the gene sequence encoding the GhGASA24 protein is shown in SEQ ID NO.

5.

4. The application of a protein or its encoding gene that improves cotton fiber length in regulating cotton fiber development, characterized in that, The protein includes one or any combination of the following: GhARF2 protein with the amino acid sequence shown in SEQ ID NO. 2, GhGASA4 protein with the amino acid sequence shown in SEQ ID NO. 4, and GhGASA24 protein with the amino acid sequence shown in SEQ ID NO. 6; The gene sequence encoding the GhARF2 protein is shown in SEQ ID NO.1; the gene sequence encoding the GhGASA4 protein is shown in SEQ ID NO.3; and the gene sequence encoding the GhGASA24 protein is shown in SEQ ID NO.

5.

5. The application of a protein or its encoding gene for improving cotton fiber length in the breeding of transgenic cotton with improved cotton fiber length, characterized in that, The protein includes one or any combination of the following: GhARF2 protein with the amino acid sequence shown in SEQ ID NO.2, GhGASA4 protein with the amino acid sequence shown in SEQ ID NO.4, and GhGASA24 protein with the amino acid sequence shown in SEQ ID NO.6; The gene sequence encoding the GhARF2 protein is shown in SEQ ID NO.1; the gene sequence encoding the GhGASA4 protein is shown in SEQ ID NO.3; and the gene sequence encoding the GhGASA24 protein is shown in SEQ ID NO.

5.

6. The application according to claim 5, characterized in that, The process of cultivating transgenic cotton with improved cotton fiber length involves introducing a vector containing the gene into cotton, inducing and screening callus tissue with upregulated protein content, and cultivating transgenic cotton that promotes cotton fiber development through plant tissue culture technology.

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