CN116284299A

CN116284299A - Protein for improving cotton fiber length and application thereof

Info

Publication number: CN116284299A
Application number: CN202310248946.9A
Authority: CN
Inventors: 陈保军; 田再垄; 何守朴; 杜雄明; 潘兆娥; 贾银华; 李洪戈; 耿晓丽; 王立如; 庞保印
Original assignee: Institute of Cotton Research of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Cotton Research of Chinese Academy of Agricultural Sciences
Priority date: 2023-03-15
Filing date: 2023-03-15
Publication date: 2023-06-23

Abstract

The invention discloses a protein for improving the length of cotton fibers and application thereof, and belongs to the field of botanics. The protein comprises one or any combination of GhARF2 protein with an amino acid sequence shown as SEQ ID NO.2, ghGAA 4 protein with an amino acid sequence shown as SEQ ID NO.4 and GhGAA 24 protein with an amino acid sequence shown as SEQ ID NO. 6. According to the invention, the functions of GhARF2, ghGAA 4 and GhGAA 24 genes in cotton fiber development are researched through molecular biological experiments, plant tissue culture techniques and the like, so that the over-expression of the genes can obviously promote the fiber development, play an important role in the fiber development, provide a theoretical basis for cotton fiber development, effectively widen cotton breeding gene resources and provide high-quality germplasm resources for cotton breeding.

Description

Protein for improving cotton fiber length and application thereof

Technical Field

The invention relates to the field of botanics, in particular to a protein for improving the length of cotton fibers and application thereof.

Background

Cotton is an important commercial crop, providing a large number of natural textile raw materials for the market. China still needs to rely on import in large quantities as cotton yield and consumption of China. Therefore, improving cotton fiber quality remains an important topic faced by cotton breeding. Cotton fibers are extremely elongated single cells formed by the differentiation and development of ovule outer skin cells. Cotton fiber development generally includes four distinct and overlapping stages: fiber initiation, fiber elongation, secondary wall thickening, and maturation. The life activities of the fibers in various periods affect the yield and quality of cotton fibers, and particularly the two periods of fiber elongation and secondary wall thickening are most closely related to the development and quality of the fibers. The specific period of fiber development is determined by the key genes, and the time-space expression specificity of the genes is mainly realized by the interaction between the transcription control factors and the promoters of the downstream genes, so that the transcription factors play an important role in the fiber development process.

ARF protein is a transcription regulatory factor critical to plant development, a gene family contains a plurality of members and mainly controls each process of plant growth and development, mainly the gene family is regulated by auxin IAA to realize the function of the gene family, cotton fiber is a single-cell structure with extremely elongated growth, no function research report on regulating cotton fiber elongation by cotton GhARF2 protein exists at present, the field needs to deeply study interaction between GhARF2 and a downstream gene promoter, and the expression of the downstream gene is regulated to further promote the biological function of fiber elongation.

Disclosure of Invention

The present invention is directed to a protein for improving the length of cotton fibers and its application to solve the above-mentioned problems of the prior art. The invention discloses that the overexpression of cotton genes GhARF2, ghGAA 4 and GhGAA 24 all promote the elongation of cotton fibers for the first time, provides a theoretical basis for the development of the cotton fibers, and effectively widens cotton breeding gene resources.

In order to achieve the above object, the present invention provides the following solutions:

the invention provides a protein for improving cotton fiber length, which comprises one or any combination of GhARF2 protein with an amino acid sequence shown as SEQ ID NO.2, ghGAA 4 protein with an amino acid sequence shown as SEQ ID NO.4 and GhGAA 24 protein with an amino acid sequence shown as SEQ ID NO. 6.

Further, the gene sequence for encoding the GhARF2 protein is shown in SEQ ID NO. 1; the gene sequence of the GhGASA4 protein is shown in SEQ ID NO. 3; the gene sequence of the GhGAA 24 protein is shown in SEQ ID NO. 5.

The invention also provides a gene for improving the length of cotton fibers, which comprises one or any combination of GhARF2 gene with a nucleotide sequence shown as SEQ ID NO.1, ghGAA 4 gene with a nucleotide sequence shown as SEQ ID NO.3 and GhGAA 24 gene with a nucleotide sequence shown as SEQ ID NO. 5.

Further, the genes also include genes which have more than 90% homology with the GhARF2, ghGAA 4 and GhGAA 24 genes and which encode GhARF2, ghGAA 4 and GhGAA 24.

The invention also provides a biological material containing the gene for improving the cotton fiber length, wherein the biological material comprises a carrier, cells and transgenic cotton.

The invention also provides a method for improving the length of cotton fibers, which comprises the step of introducing a vector containing the gene into cotton to enable the content of the protein to be up-regulated.

Further, the insertion sites of the gene GhARF2, the gene GhGASA4 and the gene GhGASA24 are included in the vector.

The invention also provides application of the protein or the gene in preparing a reagent or a combination for promoting the elongation of cotton fibers.

The invention also provides application of the protein or the gene in regulating cotton fiber development.

The invention also provides application of the protein or the gene in culturing transgenic cotton.

Further, the cultivation of the transgenic cotton comprises the steps of introducing a vector containing the gene into cotton, inducing and screening callus with up-regulated protein content, and cultivating the transgenic cotton for promoting cotton fiber development through a plant tissue culture technology.

The invention discloses the following technical effects:

the invention discloses that the overexpression of cotton genes GhARF2, ghGAA 4 and GhGAA 24 all promote the elongation of cotton fibers for the first time; comparison of the cotton bolls of GhARF2, ghasa 4 and ghasa 24 overexpressing plants with wild-type J668 revealed that the mature fibers of the GhARF2, ghasa 4 and ghasa 24 overexpressing plants were significantly larger than the wild-type fibers, respectively.

The invention discloses a cotton gene GhARF2 for the first time, which promotes the expression of downstream genes GhGAA 4 and GhGAA 24 by respectively combining promoters of GhGAA 4 and GhGAA 24 to further promote the development of cotton fibers, and is verified by Chip-Seq, yeast mono-hybrid and double luciferase assays and other biological experiments.

The invention proves that the GhARF2, ghGAA 4, ghGAA 24 and other genes regulate the length of cotton fibers and play a very important role in fiber development; overexpression of GhARF2, ghGAA 4 and GhGAA 24 significantly promoted fiber development. The function of the plant fiber in cotton fiber development is studied through molecular biology experiments, plant tissue culture technology and the like. Provides a theoretical basis for cotton fiber development, effectively widens cotton breeding gene resources, and provides high-quality germplasm resources for cotton breeding.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the expression level of GhARF2 gene in cotton tissue at different periods, wherein the transgenic local material J668 is used, 3 and 5DPA ovules and 10, 15, 20 and 25DPA fibers are respectively taken for quantitative analysis;

FIG. 2 is a graph showing statistics of fiber phenotype, fiber length and ARF2 expression levels of different transgenic cotton fibers; wherein A is the quantitative detection of ARF2, CK is 15DPA fiber of receptor material J668, ARF2-OE is 15DPA fiber which over-expresses ARF2, and three biological repeats are respectively carried out; b and C are the phenotype and fiber length statistics (bar=1 mm) of the mature fiber for controls (transgenic receptor J668, CK), ghARF2 gene overexpression (ARF 2-OE), ghARF2 gene knockout (ARF 2-Cas 9), respectively, three biological replicates;

FIG. 3 is a ChIP-Seq correlation results statistic and enrichment typing of transgenic GhARF 2; wherein A is the experimental result of cotton fiber Chip-Seq of over-expressing ARF2, which shows the sequence capable of combining with transcription factor ARF2 in genome, including the gene promoter part; b is a gene corresponding to a transcription initiation site nearest to a peak midpoint and is used for Gene Ontology (GO) analysis;

FIG. 4 shows the ability to bind to the GhGAA 4 and GhGAA 24 upstream promoters in a Chip-Seq experiment in which the GhARF2 gene is overexpressed;

FIG. 5 is a graph showing the interaction of GhARF2 with GhGAA 4 and the 2K promoter upstream of GhGAA 24 as demonstrated by yeast mono-hybrid and dual luciferase assays; wherein, A is the interaction between GhARF2 and GhGAA 4 and the upstream 2K promoter of GhGAA 24 respectively verified by a yeast single hybrid experiment; b is the interaction between GhARF2 and the upstream 2K promoter of GhGASA4 through double luciferase detection; c is the interaction between GhARF2 and the upstream 2K promoter of GhGAA 24 through double luciferase detection; D-G is a segment 5 of the upstream 2K promoter of GhGAA 4, and the dual luciferase detection verification verifies which segment of GhGAA 4 interacts with GhARF 2; H-K is a segment 5 of the upstream 2K promoter of GhGAA 24, and the dual luciferase detection verification verifies which segment of GhGAA 24 interacts with GhARF 2;

FIG. 6 is a graph showing statistics of fiber phenotypes and fiber length and GhGAA 4 and GhGAA 24 expression levels of different transgenic cotton fibers; wherein a-C are the control (transgene receptor J668, WT), the ghasa 4 gene overexpression (GASA 4 OE), and the expression level of fiber of 15DPA in cotton critical period of ghasa 4 gene interference (GASA 4 RNAi), the fiber length phenotype map and the fiber length statistical map of mature fiber (bar=1 mm), respectively; D-F are the control (transgene receptor J668, WT), ghasa 24 gene overexpression (GASA 24 OE), and fiber expression level of 15DPA during cotton key period of ghasa 24 gene interference (GASA 24 RNAi), fiber length phenotype map and fiber length statistics of mature fibers (bar=1 mm), respectively;

FIG. 7 shows the measurement and statistics of cell wall thickness and identification of cellulose content of the key cellulose related genes in different GhGAA 24 transgenic cotton; wherein A-B are respectively the expression changes of two key cellulose synthase genes in control (transgene receptors J668, WT), ghGAA 24 gene overexpression (GASA 24OE 1-3) and cotton different period fibers (5 DPA, 10DPA, 15DPA, 20 DPA) with GhGAA 24 gene interference (GASA 24 RNAi 1-3); C-G is a cell wall thickness alignment and related statistics (bar=10 μm) for controls (transgene receptor J668, WT), ghasa 24 gene overexpression (GASA 24OE 1-3), and cotton at

different periods

5, 10, 20, 30DPA of ghasa 24 gene interference (GASA 24 RNAi 1-3); h is a comparison of cellulose content measurements of cotton at

different times

5, 10, 20, 30DPA for control (transgene receptor J668, WT), ghGAA 24 gene overexpression (GASA 24OE 1-3), and GhGAA 24 gene interference (GASA 24 RNAi 1-3).

Detailed Description

The inventors have studied extensively and intensively and found for the first time a GhARF2 transcription factor capable of regulating cotton fiber/plant cell length and downstream genes GhGAA 4 and GhGAA 24. Experiments show that the over-expression of GhARF2 in cotton body promotes the elongation growth of fiber cells, and the knocking-out of the expression of GhARF2 inhibits the elongation growth of fiber cells, which proves that the GhARF2 plays a key positive regulation role in the cotton fiber elongation process. The invention discloses functions and applications of cotton GhARF2 transcription factor protein, and particularly has positive effects in promoting fiber cell elongation and improving cotton fiber length quality characteristics, and has wide application prospects.

In the course of research on cotton fiber development, the inventors cloned a gene GhARF2 (Gh_D11G 042800) encoding ARF-like protein which is specifically and highly expressed during the elongation phase of fiber cells. In order to investigate the biological function of GhARF2 in depth, the inventors performed cotton transgenic functional analysis, constructed vectors for GhARF2 overexpression and Cas9 for cotton transformation, and successfully obtained multiple transgenic lines of each vector. Analysis of transgenic plants planted in greenhouses and fields shows that overexpression of GhARF2 in cotton promotes elongation growth of fiber cells, and knockout of GhARF2 expression inhibits elongation growth of fiber cells, which indicates that GhARF2 plays a key positive regulation role in cotton fiber elongation process. Gene expression and various molecular biological assays showed that GhARF2 regulated the expression of the downstream genes GhGAA 4 (Gh_A06G 023500) and GhGAA 24 (Gh_D04G 182700). Simultaneously, the over-expression and RNA interference (RNAi) of GhGAA 4 and GhGAA 24 are respectively carried out, and the GhGAA 4 and the GhGAA 24 are found to play a positive regulation role in the cotton fiber elongation process. The results show that the transcription factors GhARF2 and the downstream genes GhGAA 4 and GhGAA 24 are important regulatory factors for cotton fiber cell development, and have great potential and application value in promoting cotton fiber elongation, improving fiber quality and the like.

The inventors have conducted the following studies with respect to cotton GhARF2 gene: the mechanism of GhARF2 regulating cotton fiber cell development is elucidated through transgenic genetic transformation and protein interaction experiments. The invention discloses information and application of GhARF2 and downstream genes GhGAA 4 and GhGAA 24 for the first time, and very unexpected discoveries that the expression quantity can be regulated to regulate the growth of cotton fiber cells, especially the over-expression of GhARF2 and the downstream genes GhGAA 4 and GhGAA 24 promote the growth of the fiber cells and increase the length of cotton fibers, and has important application value for improving the quality of cotton fibers.

The GhARF2, ghGAA 4 and GhGAA 24 genes of the invention may be in DNA form or RNA form. DNA forms include cDNA, genomic DNA, or synthetic DNA. Genomic DNA may be identical in sequence as shown in SEQ ID NO.1 or degenerate variants.

The carrier used in the examples of the present invention:

the pCAMBIA2300GFP vector was purchased from shanghai open biotechnology limited; pRGEB32 vector was purchased from Addgene; pHIS2 vectors were purchased from Pubescentis Biotechnology (Beijing) Inc.; pSK vectors were purchased from Bio-wind company; the pBI121 vector was purchased from Shanghai Kangbio-technologies Co., ltd;

pGhGAA 4-pHIS2 and pGhGAA 24-pHIS2 are achieved by single cleavage insertion of pGhGAA 4 promoter (SEQ ID NO. 23) and pGhGAA 24 promoter (SEQ ID NO. 24) at the Ecor1 site of the pHIS2 vector; ghARF2-pGADT7-Rec2 is realized by single cleavage insertion of GhARF2 (SEQ ID NO. 1) sequence at the Ecor1 site of pGADT7-Rec2 vector; 0800-ARF2 is obtained by double cleavage of pGreenII 0800 vector by Kpn1 and Pst1, insertion of GhARF2 (SEQ ID NO. 1) gene by homologous recombination, pGASA4-62SK and pGASA24-62SK by Xba1 and Spe1 on pGreenII 62-SK vector, and insertion of pGhGAA 4 promoter and pGhGAA 24 promoter by homologous recombination.

Example 1 isolation and identification of GhARF2 Gene (cDNA) sequence

1. Quantitative RT-PCR analysis of GhARF2 Gene expression

Germplasm resources: upland cotton J668 (J668 material supplied by cotton germplasm resources metaphase library of cotton institute, national academy of agricultural sciences).

Tissue samples: the J668 cotton was 3 days ovule (3 PDA ovule), 5 days ovule (5 PDA ovule), 10 days fiber (10 PDA fiber) after flowering, 15 days fiber (15 PDA fiber), 20 days fiber (20 PDA fiber) and 25 days fiber (25 PDA fiber) after flowering.

Total RNA was extracted from cotton tissue samples (performed according to Chen BJ,2021.GhGASA10-1 promotes the cell elongation in fiber development through the phytohormones IAA-reduced, BMC Plant Biology,2021,21 (1): 1-15).

2. Real-time fluorescent quantitative RT-PCR research of gene expression

The specific process is carried out with reference to He SP,2021.The genomic basis of geographic differentiation and fiber improvement in cultivated cotton,Nature Genetics,2021,53 (6): 916-924.

Taking total RNA (2 mug/sample) extracted in the step 1, and reversely transcribing the total RNA into cDNA by using M-MLVRNase HReverse Transcriptase (Promega); then, a quantitative PCR reaction was performed using cDNA as a template and using gene-specific primers (quantitative primers include GhARF 2/GhGAA 4/GhGAA 24/GhESA 10-1/GhESA 4-4, and internal reference primers) and Real-time PCR Master Mix (TOYOBO, japan), cotton GhUBQ gene as an internal standard for RT-PCR reaction, wherein the reaction procedure is: 95 degrees 30s, denaturation 95 degrees 10s, annealing 60 degrees 30s,40 cycles; the reaction system is as follows: MIX 10 μl, ddH ₂ O7. Mu.l, F/R primer 0.5. Mu.l, cDNA 2. Mu.l. Amplification of the target gene in each cycle was detected by SYBR-Green fluorescence, and specific information on the primers is shown in Table 1.

Table 1 specific sequences of each primer

As upstream transcription factors of GhASA 4 and GhASA 24, the expression level of GhASA 2 gene in the secondary wall thickening period (about 20 days after flowers) of cotton is up-regulated by real-time fluorescent quantitative RT-PCR detection through explanation of fiber expression levels of GhASA 2 in different periods (FIG. 1), so GhASA 2 is cloned from cotton, and other downstream genes have higher expression levels in fiber cell thickening. The CDS sequence of GhARF2 is obtained through DNA and protein sequence analysis, and is shown as SEQ ID NO.1, and the amino acid sequence of the protein encoded by the gene sequence is shown as SEQ ID NO. 2. CDS sequences of downstream functional genes GhGAA 4 and GhGAA 24 are shown in SEQ ID NO.3 and SEQ ID NO.5 respectively, and amino acid sequences of encoded proteins are shown in SEQ ID NO.4 and SEQ ID NO.6 respectively.

SEQ ID NO.1GhARF2 CDS sequence：

ATGACTACGTCGGAGATATCGATAAAAGGAAATTGTGTCAACGGAAGAGGAGATAGTTTTTCTTCCGGTTATACCGAGCCACGAGATACTAGGAACGCCATGGAAGGGCAGAACGGTCATTCCGCTCGTACAGCTGCCGTCAGAGAAACCGTAGACCCCGAAAGGGCGCTGTATACGGAGCTATGGCATGCATGTGCTGGACCTCTGGTGACGGTCCCTCGCGAATTAGAGCGCGTGTTCTACTTTCCTCAAGGTCACATAGAACAGGTTGAGGCGTCTACTCATCAGGTATCAGACCAGCAGATGCCGGTGTATGACCTTCCACCAAAGATCCTTTGTCGTGTGATTAACGTACAACTAAAGGCTGAACTGGATACTGATGAGGTTTTTGCTCAAGTGACTTTGCTTCCTGAACATAATCAAGATGAGAACATGGTGGACAAGGAGCCTCCCATTCTTGAACCCCCTCGGTTCCAAGTGCATTCGTTTTGCAAAACCCTGACTGCTTCAGATACGAGTACCCATGGTGGATTTTCAGTGCTCAGGCGGCATGCCGATGAATGTCTTCCACCACTGGATATGTCGCTGCAACCTCCAACACAGGAGCTGGTTTCTAAGGATTTGCATGGAAATGAGTGGCGATTCCGGCATATCTTCAGGGGTCAGCCACGAAGACACTTGCTTCAAAGCGGTTGGAGTGTTTTTGTTA

GCTCCAAGAAGCTTGTTGCTGGGGATGCATTTATATTTTTAAGAGGCGAGAATGGAGAAT

TATGCGTTGGTGTACGGCGAGCATTGAGACAACAGGGCAATGTTCCTTCATCGGTTATAT

CAAGTCATAGCATGCATCTTGGTGTGCTAGCGACAGCATGGCATGCCTACACTACCAGAA

CCATATTCACTGTGTATTACAAACCCAGGACAAGTCCAGCTGAGTTCATTGTTCCATTTAA

TCAGTACATGGAGTCGGTAAAGAACAATTACTCAATAGGGATGAGGTTCAAAATGAGAT

TTGAAGGTGAAGAAGCTCCTGAACAGAGGTTTACTGGAACAATAGTTGGAATCGAAGA

TGCTGATCCAAAAAGGTGGCAGGGTTCCAAATGGAGATGCCTGAAGGTGCGATGGGAT

GAAACGTCTACAATACCTCGTCCAGAGAGAGTTTCTCCTTGGAAAATTGAACATGCTTT

GTCTCCTCCTGCCCTTAATCCCCTTCCAATGCCCCGGCCAAAAAGGCCTCGAACTAATGC

TGTATCTTCATCCCCTGATTCCTCTGTACTTAGTAGGGAAGGTTCTTCCAAAGTTACTGTA

GACCCTTTGCCGGCCAGTTCATTTTCAAGGGTCTTGCAAGGTCAAGAATTCTCGACCTT

GAGAGGCACATTTGCTGAGAGTAATGATTCTGAAACTGCTGATAGGTCAGTGATGTGGC

CACCTTCAATAGATGATGAGAAGATTGATGTAGCTCATGGTGAAAGAAAATTTGGGTCA

GAGAATTGGATGCCCTCTAGGAGGCATGAACCAACTTACACAGATTTGCTCTCAGGTTTT

GGGTCGAATGCTGATACATCGCGCGGATATTATCCTTCCTTTGTTGATCAAACTTCAGTAG

CTGGTAATTCGGGGAAAAAACAATTACTAGGTCAAGAAGGGAAGCTTGGCTCTTGGTCC

CTCCTGCCATCTGGTCTCTCACTCAAGTTGTCTGACAGTAGTACAGACCCTCCTTTGCAA

GGTTCTGATGTGCCTTGTCAGGCGCGGGGAAATGGTAGATTTAGTGGTTTTGGTGACTAC

CCTATACTTGAAGGTCGTAGGATTGAATGCTCACGTGGTAATTGGTTGATGCCTCCCCCA

ACCACTTCTTGTTATGATAATTCAATCCAGTCAAGAGATTTAATGCCGAAAACATCATTGG

CTCAAGAGCATAAGAATGGAAAATCTAGAGAAGGAAACTGCAAGCTCTTTGGTATTCCT

CTCATAAGTGCTTCTAGCGCATCAGAGCCTGCAGTCTCTCATATTAGTGCTTTCGCCAAG

CCTGTAGGACATATGCAAGCTGCATTGCACCAGGTTCATGCACTTGAATCTGATAAAAGG

TCTGAAAATTCAAACGCCTCCCAGATGGCAGAGGATGTTTCTGCTTTTAATGAGCAGGA

GAAAATAGTGAAGCTGGGTCAGCCCCATGCACGGGAGTTTCAAAGCAAACTGTCTACT

GCTTCAACTAGGAGTTGTACTAAGGTTCTCATGCAGGGGACTGCTCTTGGAAGGTCTGT

GGACCTTACCAAGTTCAACAACTATGATGAGTTGATCGCTGAATTGGATCAATTATTTGA

GTTTGGAGGTGAATTAATGGCCCCTCAAAAGAACTGGCTTGTTGTTTATACTGATGATGA

GGGTGATATGATGCTTGTTGGCGATGATCCTTGGCAGGAATTTTGTGCCATGGTCCGCAA

GATTGGTATCTACACTAGGGAAGAGGTCCAGAAGATGAAGCCAGGGTCGTTGGGTTCAA

AGTTTGAGGACATTCCAGTTCCCACAGAAGGTACAGTTGCAAAAGAAGTGAACTGTCC ATCAGCATCTAGTGCAAAGAATTGTTCAGGGTAA。

SEQ ID NO.2 GhARF2 protein sequence：

MTTSEISIKGNCVNGRGDSFSSGYTEPRDTRNAMEGQNGHSARTAAVRETVDPERALYTELWHACAGPLVTVPRELERVFYFPQGHIEQVEASTHQVSDQQMPVYDLPPKILCRVINVQLKAELDTDEVFAQVTLLPEHNQDENMVDKEPPILEPPRFQVHSFCKTLTASDTSTHGGFSVLRRHADECLPPLDMSLQPPTQELVSKDLHGNEWRFRHIFRGQPRRHLLQSGWSVFVSSKKLVAGDAFIFLRGENGELCVGVRRALRQQGNVPSSVISSHSMHLGVLATAWHAYTTRTIFTVYYKPRTSPAEFIVPFNQYMESVKNNYSIGMRFKMRFEGEEAPEQRFTGTIVGIEDADPKRWQGSKWRCLKVRWDETSTIPRPERVSPWKIEHALSPPALNPLPMPRPKRPRTNAVSSSPDSSVLSREGSSKVTVDPLPASSFSRVLQGQEFSTLRGTFAESNDSETADRSVMWPPSIDDEKIDVAHGERKFGSENWMPSRRHEPTYTDLLSGFGSNADTSRGYYPSFVDQTSVAGNSGKKQLLGQEGKLGSWSLLPSGLSLKLSDSSTDPPLQGSDVPCQARGNGRFSGFGDYPILEGRRIECSRGNWLMPPPTTSCYDNSIQSRDLMPKTSLAQEHKNGKSREGNCKLFGIPLISASSASEPAVSHISAFAKPVGHMQAALHQVHALESDKRSENSNASQMAEDVSAFNEQEKIVKLGQPHAREFQSKLSTASTRSCTKVLMQGTALGRSVDLTKFNNYDELIAELDQLFEFGGELMAPQKNWLVVYTDDEGDMMLVGDDPWQEFCAMVRKIGIYTREEVQKMKPGSLGSKFEDIPVPTEGTVAKEVNCPSASSAKNCSG。

SEQ ID NO.3 GhGASA4 CDS sequence：

ATGAAGATGGTATTGGTGCTTTTCTTGCTTGTTTCTCTTGCTCTCAGCTCTTGTTTCTTCGAGGTGTCGATTGCCGGTTCGGATTTTTGTGACTCAAAGTGTGCGGTGAGGTGCTCAAAGGCAGGGGTTCAAGACAGGTGTTTGAAATATTGTGGGATTTGTTGTGAGAAATGTCATTGTGTTCCATCTGGGACATTTGGGCATAAAGATGAATGCCCTTGTTATAGGGACATGAAGAACTCTAAGGGCAAATCCAAGTGCCCTTAG。

SEQ ID NO.4 GhGASA4 protein sequence：

MKMVLVLFLLVSLALSSCFFEVSIAGSDFCDSKCAVRCSKAGVQDRCLKYCGICCEKCHCVPSGTFGHKD ECPCYRDMKN SKGKSKCP。

SEQ ID NO.5 GhGASA24 CDS sequence：

ATGAAGCTCTTGTTTCTAACTTTGCTGCTTTGTTCTCTTCTTCTATGTTCTTCAGTTTTTGCACCAACAATGGCTCAGCCTCGTTCACCTTTTTGTGAAGGGAAATGCAAAGGGAGGTGCAATAAAGCGGCGGTTTGGGATCGGTGCTTCAAATATTGCGGCATATGTTGCGAGGAGTGTCAATGCGTTCCGTCCGGTACTTACGGGAACAAACACGAGTGTCCTTGCTACAGAGAT AAGGTGAACAACAAGGGCAAACCCAAATGCCCTTGA。

SEQ ID NO.6GhGASA24 protein sequence：

MKLLFLTLLLCSLLLCSSVFAPTMAQPRSPFCEGKCKGRCNKAAVWDRCFKYCGICCE ECQCVPSGTYGNKHECPCYRDKVNNKGKPKCP。

EXAMPLE 2 construction of GhARF2 transgenic cotton and fiber Length statistics

1. Construction of plant expression vectors

The overexpression vector p2300-GhARF2 of the GhARF2 gene and the vector pRGEB32-GhU6.7-NPT2-GhARF2 for knocking out the GhARF2 gene are respectively constructed.

The over-expression vector p2300-GhARF2 is subjected to double enzyme digestion by the pCAMBIA2300GFP vector at BamH I and Sac I sites, and after GFP labels are cut off, a homologous recombination method is used for connecting a sequence shown in SEQ ID NO.1 to the vector;

vector pRGEB32-GhU6.7-NPT2-GhARF2 for knocking out GhARF2 is obtained by connecting a tRNA-PAM1-gRNA-tRNA-PAM2-gRNA sequence (SEQ ID NO. 22) with double targets to pRGEB32 vector driven by ubiquitin U6.7 by using CRISPR/Cas9 gene editing technology and using a homologous recombination method to knock out the sequence shown in SEQ ID NO. 7.

Specific construction methods refer to Tian ZL,2022.Strigolactones act downstream of gibberellins toregulate fiber cell elongation and cell wall thickness in cotton (Gossypium hirsutum).

SEQ ID NO.7GhARF2 knock out sequence:

GAACCCCCTCGGTTCCAAGTGCATTCGTTTTGCAAAACCCTGACTGCTTCAGATACGAGTACCCATGGTGGATTTTCAGTGCTCAGGCGGCATGCCGATGAATGTCTTCCACCACTGGATATGTCGCTGCAACCTCCAACACAGGAGCTGGTTTCTAAGGATTTGCATGGAAATGAGTGGCGATTCCGGCATATCTTCAGGGGTCAGCCACGAAGACACTTGCTTCAAAGCGGTTGGAGTGTTTTTGTTAGCTCCAAGAAGCTTGTTGCTGGGGATGCATTTATATTTTTAAGAGGCGAGAATGGAGAATTATGCGTTGGTGTACGGCGAG。

SEQ ID NO.22：

AACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCACCCCTCGGTTCCAAGTGCATTCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCAGAGAATTATGCGTTGGTGTACGGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC。

2. Construction of transgenic plants

The constructed p2300-GhARF2 vector and pRGEB32-GhU6.7-NPT2-GhARF2 vector are respectively transferred into LBA4404 agrobacterium by an electrotransformation method, cotton hypocotyl explants are transfected by using upland cotton J668 as a receptor material, after 2 days of co-culture, the hypocotyls are transferred to a selective medium for culture, and the transformed callus is induced and screened. And (3) performing subculture on the selected callus for 8-10 months, and inducing differentiation to obtain embryogenic callus and somatic embryos, wherein the somatic embryos further germinate and regenerate transgenic cotton seedlings. Transplanting cotton seedlings into soil, and growing until the cotton seedlings bloom and fruit. Extracting cotton genome DNA, and identifying transgenic cotton plants by using a PCR technology through agarose gel electrophoresis band detection (the knocked-out transgenic plants do not contain GhARF2 genes and further do not have corresponding electrophoresis bands, and the overexpressed transgenic plants pass through cotton 35S strong promoter primer (GACGCACAATCCCACTATCC) and gene self primer GhARF2-R detection).

3. Transgenic plant cotton fiber length statistics

Over-expressed and knockout transgenic lines of more than 5T 0 generation cotton GhARF2 genes were obtained, and fiber lengths during maturity of T3 generation cotton plants were photographed and statistically analyzed (B and C of fig. 2) and expression amounts of GhARF2 in fibers during key periods (a of fig. 2) by two seed generations, respectively. The results show that high expression of GhARF2 promotes fiber length elongation.

EXAMPLE 3 Chip-Seq sequencing of GhARF2 transgenic Cotton fibers

Chip-Seq sequencing step:

(1) Formaldehyde cross-links the whole cell line (fiber), i.e. links the target protein to chromatin;

(2) Isolating genomic DNA and breaking it into small fragments of a certain length by ultrasound;

(3) Adding an antibody specific for the protein of interest, the antibody forming an immunoprecipitated immunobinding complex with the protein of interest;

(4) Removing cross-linking, purifying DNA to obtain a DNA sample of chromatin immunoprecipitation, and preparing for sequencing;

(5) The prepared samples were deep sequenced.

To see which downstream genes are directly under GhARF2 regulation, chromatin immunoprecipitation sequencing (ChIP-Seq) was performed. We Immunoprecipitated (IP) protein-DNA complexes in 15DPA transgenic fibers and degraded ARF2 protein using Flag antibodies and protein G MagBeads. We sequenced two IPs and one control library. In the IP experiment, there were 2509 peaks on the chromosome (a of fig. 3). To further explore the binding site characteristics of protein modifications, we found the gene corresponding to the transcription initiation site nearest to the peak midpoint, knowing the regulatory mechanism of protein modifications on the gene, for Gene Ontology (GO) analysis (B of fig. 3). These genes are involved in cell wall formation, phytohormone response and energy production, and are involved in fiber development. We also calculated the number of functional elements per gene distributed over the genome. About 7.69% of the peak was located at the promoter of the gene. Transcriptional profiling showed that 28 candidate genes were strongly expressed during cotton fiber elongation and secondary cell wall thickness. The promoter and gene analysis of the above promoters respectively show that the promoter sites of the two genes GhGAA 4 and GhGAA 24 have peaks (figure 4), and the two genes are specifically expressed in fibers, so that the expression quantity of GhARF2 is predicted to be regulated by combining the promoters of the two genes to further regulate the elongation of cotton fibers.

Example 4 Yeast Monohexaction and LUC experiments

First, pGhGAA 4-pHIS2, pGhGAA 24-pHIS2 and the gene GhARF2-pGADT7-Rec2 were constructed with the promoter 2000bp, and the optimum concentration of 150mM was selected by co-transferring yeast Y187 and later by 3-AT, and then pGhGAA 4-pHIS2 and GhARF2-pGADT7-Rec2 interaction was verified in yeast, and pGhGAA 24-pHIS2 and GhARF2-pGADT7-Rec2 interaction (FIG. 5A). The 0800-ARF2, pGASA4-62sk and pGASA24-62sk vectors were then constructed and transferred into Agrobacterium LBA4404, respectively, and co-transferred into tobacco, 0800-ARF2 and pGASA4-62sk were fluorogenic (B of FIG. 5), 0800-ARF2 and pGASA24-62sk were fluorogenic (C of FIG. 5). To further verify which binding element of pGhGASA4 and pGhGASA24 is capable of interacting with the transcription factor ARF2, pGhGASA4 and pGhGASA24 promoters were segmented into 5 segments, respectively, and pHis2 vectors and 62sk vectors were constructed, respectively, to further verify which portions of their promoters interacted with the transcription factor, both methods together verified pGASA4P4 (D-G of fig. 5) and pGASA24P3 (H-K of fig. 5) interacted with the transcription factor ARF2. Analysis found that there was this binding element TGTCTC on both pGASA4P4 and pGASA24P3 promoters, indicating that ARF2 further regulates expression of ghasa 4 and ghasa 24 by binding to the binding element TGTCTC in pGhGASA4 and pGhGASA24, further regulating cotton fiber length.

EXAMPLE 5 construction of GhGAA 4 and GhGAA 24 transgenic cotton and fiber Length statistics

Construction of GhGAA 4 (SEQ ID NO. 3) and GhGAA 24 (SEQ ID NO. 5) overexpression vectors p 2300-GhGAA 4 and p 2300-GhGAA 24, and plant expression vectors of GhGAA 4-RNAi sequence (SEQ ID NO. 8) and GhGAA 24-RNAi sequence (SEQ ID NO. 9).

The construction method of the p 2300-GhGAA 4 and p 2300-GhGAA 24 vector comprises the following steps:

the pCAMBIA2300GFP vector was digested at BamHI and SacI sites, and after cleavage of GFP tag, the coding sequences of GhGAA 4 and GhGAA 24 (shown in SEQ ID NO.3 and SEQ ID NO. 5) were ligated to the vector, respectively, using a homologous recombination method.

The construction method of the vector containing GhGAA 4-RNAi and GhGAA 24-RNAi sequences comprises the following steps:

for RNAi systems, 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 about 300bp (SEQ ID NOS.8-9) of GhGAA 4 and GhGGSAS 24 was ligated before the TUB1 intron, and the corresponding antisense strand was ligated after the TUB1 intron, to form the coding strand-intron-antisense strand recombination sequence. This sequence was ligated between BamH 1 and Sac I sites of pBI121 vector by homologous recombination method, and GUS tag of pBI121 vector was replaced.

SEQ ID NO.8GhGASA4 RNAi sequence:

TGGTGCTTTTCTTGCTTGTTTCTCTTGCTCTCAGCTCTTGTTTCTTCGAGGTGTCGAT TGCCGGTTCGGATTTTTGTGACTCAAAGTGTGCGGTGAGGTGCTCAAAGGCAGGGGTTC AAGACAGGTGTTTGAAATATTGTGGGAT。

SEQ ID NO.9GhGASA24 RNAi sequence:

GCTCTTGTTTCTAACTTTGCTGCTTTGTTCTCTTCTTCTATGTTCTTCAGTTTTTGCAC CAACAATGGCTCAGCCTCGTTCACCTTTTTGTGAAGGGAAATGCAAAGGGAGGTGCAAT AAAGCGGCGGTTTGGGATCGGTGCTTCAAATATTGCGGCATATGTTGCGAG。

SEQ ID NO.25：

GTAATTTTGGTTAATTTAAAGGTTCCATTTGAAGAGTTAAGTCCGGTTTATCTTTGAA TTGAGCCTCTGTTTGGTTACAG。

Transferring the constructed vector into LBA4404 agrobacterium by an electrotransformation method, and infecting the agrobacterium with cotton hypocotyl to obtain transgenic cotton (the method is as above). Transgenic cotton was identified by DNA extraction and PCR amplification, and the expression levels of ghasa 4 (a of fig. 6) and ghasa 24 (D of fig. 6) in T3-generation cotton maturity fiber length and key 15DPA fibers were counted. Photographic and statistical results of the mature fibers showed that ghasa 4 (B and C of fig. 6) and ghasa 24 (E and F of fig. 6) promote fiber length elongation, respectively. In the study it was found that GhGAA 24 might promote fibroblast wall synthesis, and qPCR quantitative results showed that GhGAA 24 promoted expression of cellulose synthase GhCas 10-1 (A of FIG. 7) and GhCas 4-4 (B of FIG. 7). From paraffin sections (experimental method 1 below) and cellulose detection (method 2 below) analysis (C-H of FIG. 7) of fibers 5DPA, 10DPA, 20DPA and 30DPA at different times, it was found that GhGAA 24 promoted cell wall thickening and increased cellulose synthesis in 20DPA and 30 DPA.

1. Paraffin section: 5. cotton fibers of 10, 20, 30DPA were fixed with FAA solution and dehydrated in ethanol series. The fiber ends closest to the ovule were embedded in paraffin wax with ceresin and cut into 10 μm thick cross sections according to the published method (Huang et al, 2021) with a rotary microtome (Reichert-Histo stat, germany). Sections were observed under confocal laser scanning microscopy (LSM 710, zeiss, germany).

2. Cellulose extraction and measurement was performed using a cellulose assay kit (Solarbio, china). The fiber sample (0.1 g) was frozen in liquid nitrogen and lyophilized, 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 ℃ for 20 minutes. After cooling to room temperature, centrifugation was carried out at 6000g for 10 minutes. The particles were then washed twice, first with 80% ethanol and then with acetone. An appropriate amount of amylase was then added to remove starch, the sample was centrifuged at 6000g for 20 minutes and the pellet was dried at 50 ℃. The pellets are respectively treated with H ₂ O and H ₂ SO ₄ Dissolve and centrifuge for 10 minutes. Then use H ₂ O the supernatant was diluted, a working solution was added, and the cellulose content was calculated by measuring absorbance at 620 nm.

The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims

1. A protein for improving cotton fiber length, which is characterized by comprising one or any combination of GhARF2 protein with an amino acid sequence shown as SEQ ID NO.2, ghGAA 4 protein with an amino acid sequence shown as SEQ ID NO.4 and GhGAA 24 protein with an amino acid sequence shown as SEQ ID NO. 6.

2. The protein of claim 1, wherein the gene sequence encoding the GhARF2 protein is set forth in SEQ ID No. 1; the gene sequence of the GhGASA4 protein is shown in SEQ ID NO. 3; the gene sequence of the GhGAA 24 protein is shown in SEQ ID NO. 5.

3. A gene for improving cotton fiber length, which is characterized in that the gene comprises one or any combination of GhARF2 gene with a nucleotide sequence shown as SEQ ID NO.1, ghGAA 4 gene with a nucleotide sequence shown as SEQ ID NO.3 and GhGAA 24 gene with a nucleotide sequence shown as SEQ ID NO. 5.

4. A biological material comprising the gene for improving cotton fiber length of claim 3, wherein said biological material comprises a vector, a cell, and transgenic cotton.

5. A method for improving the length of cotton fibers, characterized in that a vector comprising the gene of claim 3 is introduced into cotton to up-regulate the protein content.

6. The method of claim 5, wherein the vector comprises insertion sites for gene GhARF2, gene GhGAA 4 and gene GhGAA 24.

7. Use of a protein according to any one of claims 1-2 or a gene according to claim 3 for the preparation of an agent or combination that promotes elongation of cotton fibers.

8. Use of a protein according to any one of claims 1-2 or a gene according to claim 3 for modulating cotton fiber development.

9. Use of a protein according to any one of claims 1-2 or a gene according to claim 3 for the cultivation of transgenic cotton.

10. The use according to claim 9, wherein said growing transgenic cotton comprises introducing a vector containing said gene into cotton, inducing and selecting callus tissue with up-regulated protein content, and growing transgenic cotton promoting cotton fiber development by plant tissue culture techniques.