WO2018010513A1 - Cotton ghtcp4 gene and application thereof in modifying length of cotton fiber - Google Patents

Abstract

A cotton GhTCP4 gene and an application thereof in modifying the length of a cotton fiber. The cotton GhTCP4 gene is isolated, cloned and subjected to functional identification, and an expression level of the GhTCP4 gene is regulated by molecular biology and transgenic technology, so as to make adjustment to plant growth and development, thereby obtaining a transgenic cotton having significantly longer fibers. The function and use of a GhTCP4 transcriptional factor-like protein in cotton have a positive effect on promoting the elongation of fibroblasts and improving the length and quality of cotton fibers, and thus have a broad application prospect.

Description

Cotton GhTCP4 gene and its application in improving cotton fiber length Technical field

The present invention relates to the field of botany, and more particularly to the cotton GhTCP4 gene and its use in improving cotton fiber length.

Background technique

Cotton is an important economic crop that provides the market with a large amount of natural textile raw materials. China is the world's largest cotton-producing country and consumer country, but the high-quality raw cotton of high-grade cotton yarn is mostly imported, and the medium and long-staple quality raw cotton suitable for spinning 60-120 yarns is lacking. Therefore, improving cotton fiber quality is an important issue for cotton breeding. Cotton fiber is a single cell developed by the epidermal layer of cotton ovule. Its differentiation and development process can be divided into four stages: fiber blast cell differentiation and protrusion, rapid elongation of fiber cells, secondary wall synthesis and dehydration maturation. The life activities of fiber cells affect the yield and quality of cotton fiber. The two stages of fiber elongation and secondary wall synthesis are most closely related to fiber development and quality formation. Cotton fiber development and structure are characterized by a series of genes expressed at specific time and space, and the temporal and spatial specificity of gene expression is mainly achieved by the interaction between transcriptional regulators and promoters of downstream genes. Therefore, transcription factors play an important role in controlling the initiation and development of cotton fibers.

The TCP protein is a plant-specific transcriptional regulator. The gene family contains multiple members. Its main function is to control the morphogenesis of leaves and flowers. This effect is often achieved by regulating the division and differentiation of cells. Cotton fiber is a kind of single-cell structure with extremely elongated growth. There is no research report on the function of cotton GhTCP protein to regulate cotton fiber cell development. The biological function of GhTCP needs to be further studied in this field.

Summary of the invention

It is an object of the present invention to provide a cotton GhTCP4 gene and its use in improving the length of cotton fibers.

In a first aspect of the invention, there is provided a use of a GhTCP4 gene or a protein encoded thereby for one or more uses selected from the group consisting of:

(a) for the preparation of an agent or composition that promotes elongation of a plant cell;

(b) for promoting elongation of plant cells;

(c) for the preparation of a reagent or composition for promoting elongation of cotton fibers;

(d) Used to promote elongation of cotton fibers.

In another preferred embodiment, the plant cell is a fibroblast.

In another preferred embodiment, the plant is selected from the group consisting of Malvaceae, Solanaceae, and Cruciferae.

In another preferred embodiment, the plant is selected from the group consisting of cotton, tobacco, and Arabidopsis.

In another preferred embodiment, the plant cell is a cotton fiber cell.

In another preferred embodiment, the GhTCP4 gene comprises a wild-type GhTCP4 gene and a mutant GhTCP4 gene.

In another preferred embodiment, the mutant comprises a mutant form in which the function of the encoded protein is not altered (i.e., the function is identical or substantially identical to the wild-type encoded protein).

In another preferred embodiment, the polypeptide encoded by the mutant GhTCP4 gene is identical or substantially identical to the polypeptide encoded by the wild-type GhTCP4 gene.

In another preferred embodiment, the mutant GhTCP4 gene comprises a polynucleotide having a homology of ≥ 80% (preferably ≥ 90%, more preferably ≥ 95%) compared to the wild-type GhTCP4 gene.

In another preferred embodiment, the mutant GhTCP4 gene comprises a truncation or addition of 1-60 (preferably 1-30, more preferably 1) at the 5' end and/or the 3' end of the wild type GhTCP4 gene. -10) nucleotide polynucleotides.

In another preferred embodiment, the GhTCP4 gene comprises the A subgroup GhTCP4 gene and the D subgroup GhTCP4 gene, preferably the D subgroup GhTCP4 gene.

In another preferred embodiment, the gene comprises genomic DNA, cDNA, and/or mRNA.

In another preferred embodiment, the CDS sequence of the GhTCP4 gene (subgroup D) is set forth in SEQ ID NO.: 1.

In another preferred embodiment, the (predicted, subgroup D) encoded protein of the GhTCP4 gene is set forth in SEQ ID NO.: 2.

In another preferred embodiment, the genomic sequence of the GhTCP4 gene (subgroup D) is set forth in SEQ ID NO.: 3.

In another preferred embodiment, the (predicted, subgroup A) encoded protein of the GhTCP4 gene is set forth in SEQ ID NO.: 23.

In another preferred embodiment, the GhTCP4 gene is derived from a plant, preferably from a Malvaceae plant, more preferably from cotton, and is most preferably derived from upland cotton.

In another preferred embodiment, the GhTCP4 gene is a gene derived from the A subgroup or the D subgroup of the Upland cotton.

In a second aspect of the invention, a method of regulating the length of a plant cell is provided, comprising the steps of:

(a) introducing a foreign construct into a plant cell, wherein the construct contains an exogenous GhTCP4 gene sequence, an exogenous nucleotide sequence that promotes expression of the GhTCP4 gene, or an exogenous nucleotide that inhibits expression of the GhTCP4 gene. a sequence to obtain a plant cell into which the exogenous construct is introduced;

(b) the plant cells introduced into the exogenous construct obtained in the previous step are regenerated into plants: and

(c) optionally identifying the regenerated plant to obtain a plant having a change in plant cell length.

In another preferred embodiment, the plant having a plant cell length change refers to a change in plant cell length as compared to the parent plant.

In another preferred embodiment, the exogenous GhTCP4 gene sequence further comprises a promoter and/or terminator operably linked to the ORF sequence.

In another preferred embodiment, the promoter is selected from the group consisting of a constitutive promoter, a tissue-specific promoter, an inducible promoter, and a strong promoter.

In another preferred embodiment, the constitutive promoter comprises a 35S promoter.

In another preferred embodiment, the exogenous nucleotide sequence comprises a nucleotide sequence that interferes with expression of the GhTCP4 gene.

In another preferred embodiment, the exogenous nucleotide sequence comprises an RNA interference sequence.

In a third aspect of the invention, there is provided a method of promoting elongation of cotton fibers, the method comprising the steps of inhibiting expression of a GhTCP4 gene or inhibiting activity of a GhTCP4 protein in the plant.

In another preferred embodiment, said promoting cotton fiber elongation comprises promoting elongation of cotton fiber cells.

In another preferred embodiment, the method comprises administering an inhibitor of the plant GhTCP4 gene or a polypeptide encoded thereby.

In another preferred embodiment, the method comprises introducing into the plant an exogenous nucleotide sequence that inhibits expression of the GhTCP4 gene.

In another preferred embodiment, the exogenous nucleotide sequence comprises an RNAi interference sequence.

In another preferred embodiment, the method comprises the steps of:

(a) Agrobacterium providing an expression vector carrying the GiTCP4 gene RNAi interference sequence;

(b) contacting the plant cell or tissue or organ with the Agrobacterium in step (a), thereby transferring the GhTCP4 gene RNAi interference sequence into the plant cell and integrating it into the chromosome of the plant cell;

(c) selecting a plant cell or tissue or organ that has been transferred into the GhTCP4 gene RNAi interference sequence;

(d) regenerating the plant cell or tissue or organ in step (c) into a plant.

In another preferred embodiment, the RNAi interference sequence is set forth in SEQ ID NO.: 4.

In a fourth aspect of the invention, there is provided a use of a modulator of the GhTCP4 gene or a protein encoded thereby for one or more uses selected from the group consisting of:

(a) an agent or composition for preparing a plant cell length;

(b) for regulating the length of plant cells;

(c) an agent or composition for preparing a cotton fiber length; and

(d) used to regulate the length of cotton fibers.

In another preferred embodiment, the composition comprises an agricultural composition.

In another preferred embodiment, the modulator comprises an accelerator, an inhibitor.

In another preferred embodiment, the modulator is an inhibitor, and the regulation refers to promoting elongation of plant cells and/or cotton fibers.

In another preferred embodiment, the modulator is an accelerator, and the regulation refers to inhibiting elongation of plant cells and/or cotton fibers.

In another preferred embodiment, the modulator comprises a small molecule compound, or a nucleic acid substance.

In another preferred embodiment, the nucleic acid species is selected from the group consisting of miRNA, shRNA, siRNA, or a combination thereof.

In a fifth aspect of the invention, there is provided a transgenic plant into which a GhTCP4 gene is introduced.

In another preferred embodiment, the transgenic plant is a transgenic cotton plant.

In another preferred embodiment, the transgenic plants are used to promote cotton fiber cell growth, increase cotton fiber length, and/or improve cotton fiber quality.

In a sixth aspect of the invention, there is provided a cotton fiber length-related polypeptide having a function of regulating the length of cotton fibers, and wherein the polypeptide is selected from the group consisting of:

(i) a polypeptide of the amino acid sequence of SEQ ID NO.: 2;

(ii) a polypeptide having the function of regulating cotton fiber length formed by substituting, deleting or adding a polypeptide represented by (i) by one or several amino acid residues;

(iii) a polypeptide having an amino acid sequence having a homology of any of the polypeptides shown in (i) ≥ 95% (preferably ≥ 98%, more preferably ≥ 99%) having a function of regulating cotton fiber length.

In a seventh aspect of the invention, a gene encoding the cotton fiber length-related polypeptide of the sixth aspect of the invention is provided.

In an eighth aspect of the invention, a vector comprising the gene of the seventh aspect of the invention is provided.

In a ninth aspect of the invention, there is provided a genetically engineered host cell comprising the vector of the eighth aspect of the invention or the gene of the seventh aspect of the invention integrated in the genome of the invention.

In another preferred embodiment, the host cell is selected from the group consisting of a plant cell, a prokaryotic cell.

In another preferred embodiment, the host cell comprises a Malvaceae plant cell.

In another preferred embodiment, the host cell comprises a cotton plant cell.

In a tenth aspect of the invention, a method of preparing the polypeptide of the sixth aspect of the invention, the method comprising:

(a) cultivating the host cell of the ninth aspect of the invention under conditions suitable for expression;

(b) isolating the polypeptide of the sixth aspect of the invention from the culture.

It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here.

DRAWINGS

Figure 1 shows the sequence analysis of upland cotton GhTCP4.

Figure 1A shows the homologous alignment of the protein sequences of GhTCP4A, GhTCP4D and Arabidopsis TCP4 in the two subgroups of Upland cotton. Among them, the red underline shows the TCP domain conserved by the TCP protein.

Figure 1B shows the phylogenetic tree of GhTCP4 and the Arabidopsis TCP protein family constructed using MEGA software, using the maximum likelihood method.

Figure 1C shows the results of the homology alignment of the representative gene of the cotton genus TCP4. Protein sequence homology was generated by BioEdit software.

Figure 2 shows the analysis of GhTCP4 expression characteristics.

Fig. 2A shows the results of detecting the expression level of GhTCP4 in developing fibroblasts.

Figure 2B shows the relative expression levels of GhTCP4A and GhTCP4D in different developmental stages of fibroblasts using the pyrophosphate genotyping technique. Among them, black represents the relative expression level of GhTCP4A, and green (gray) represents the relative expression amount of GhTCP4D. The number of days after flowering is indicated by DPA (days post anthesis); 0DPA is the ovule and the rest are fibroblasts.

Figure 2C shows the expression characteristics of GbTCP4 in different fiber developmental stages of sea-island cotton.

Figure 2D shows the expression characteristics of GaTCP4 in different fiber developmental stages of Asian cotton.

Figure 3 shows a functional analysis of GhTCP4 regulation of leaf development.

Figure 3A shows that GhTCP4 overexpressing anti-miR319 cleavage in cotton results in leaf phenotypic changes.

Figure 3B inhibits GhTCP4 expression in cotton resulting in leaf phenotypic changes.

Figure 3C shows the subcellular localization of GhTCP4-GFP in tobacco epidermal cells. Among them, the nucleus was visualized by DAPI staining.

Figure 4 shows fiber phenotypic analysis of 35S::dsGhTCP4 transgenic cotton (ds represents double-stranded RNA interference, abbreviated as double-stranded RNA interference). The error line is shown as the standard deviation (STDEV); "***" indicates that the experimental group and the control group were subjected to the two-sample equal variance hypothesis, Student's t-test analysis, P < 0.001, n = 30.

Figure 4A shows the amount of GhTCP4 expression in the fibers detected by qRT-PCR.

Figure 4B shows the variable length phenotype of the 35S::dsGhTCP4 transgenic cotton fiber.

Figure 4C shows the fiber length statistics for 35S::dsGhTCP4 transgenic cotton.

Figure 5 shows fiber phenotypic analysis of RDL1::mGhTCP4 transgenic cotton (OE stands for overexpression, an abbreviation for Over-expression). The error line is shown as the standard deviation (STDEV); "***" indicates that the experimental group and the control group were subjected to the two-sample equal variance hypothesis, Student's t-test analysis, P < 0.001, n = 30.

Figure 5A shows the detection of the amount of GhTCP4 expression in fibroblasts at 6 and 9 days after flowering.

Figure 5B shows a short phenotype of RDL1::mGhTCP4 transgenic cotton fiber, Bar = 1 cm.

Figure 5C shows fiber length statistics for different strains of RDL1::mGhTCP4 transgenic cotton.

Figure 6 shows the interaction of GhTCP4 with GhHOX3.

Figure 6A shows that GhTCP4 is capable of binding to the full length of GhHOX3 and to portions containing both the Leu-zipper domain and the START domain.

Figure 6B shows that GhTCP4 interacts with GhHOX3 based on a half-molecule fluorescence complementation assay based on firefly luciferase; a strong fluorescence signal can be observed in the experimental group compared to the control, indicating that GhTCP4 interacts with GhHOX3.

Figure 6C shows that the co-immunoprecipitation experiment demonstrated that GhTCP4 interacts with GhHOX3.

Figure 7 shows that GhTCP4 interacts with GhHOX3 to inhibit GhHOX3 activation of downstream genes.

Figure 7A shows that GhTCP4 inhibits the activation of GhRDL1 promoter by GhHOX3.

Figure 7B shows that GhTCP4 inhibits the activation of the GhEXPA1 promoter by GhHOX3.

Figure 7C shows that GhTCP4 inhibits the activation of the GhRDL1 promoter by GhHOX3-GhHD1 heterodimer.

Figure 7D shows that GhTCP4 inhibits the activation of the GhEXPA1 promoter by GhHOX3-GhHD1 heterodimer.

Figure 8 shows a schematic diagram of the principle of site-directed mutagenesis GhTCP4. Among them, P1(S) and P2(AS) are the full-length sequences of the fragment to be mutated. P3 (AS) is the reverse primer upstream of the mutation site. The 5' end of the P4 primer is 15-20 bp complementary to the primer P3, and the 3' end is about 20 bp complementary to the sequence downstream of the mutation site, and the light color in the middle is shown as the mutated sequence.

detailed description

The inventors have extensively and intensively studied, and for the first time, unexpectedly discovered a GhTCP4 gene capable of regulating the length of cotton fiber/plant cells. Experiments showed that overexpression of GhTCP4 in cotton inhibited the elongation of fibroblasts, while inhibition of GhTCP4 expression promoted the elongation of fibroblasts, indicating that GhTCP4 plays a key negative regulatory role in cotton fiber elongation. The invention discloses the function and use of the cotton GhTCP4 transcription factor-like protein, and has an active role in promoting fiber cell elongation and improving cotton fiber length quality traits, and has broad application prospects.

the term

As used herein, the term "specific expression" refers to the expression of a gene of interest at a particular time in a plant and/or the expression of a particular tissue.

As used herein, "exogenous" or "heterologous" refers to the relationship between two or more nucleic acid or protein sequences from different sources. For example, if the combination of a promoter and a gene sequence of interest is generally not naturally occurring, the promoter is foreign to the gene of interest. A particular sequence is "exogenous" to the cell or organism into which it is inserted.

GhTCP4 gene

As used herein, the terms "GhTCP4 gene", "cotton fiber length-related gene", and "gene of the present invention" are used interchangeably and refer to a gene of the present invention having a cotton fiber length.

The TCP protein is a plant-specific transcriptional regulator. The gene family contains multiple members. Its main function is to control the morphogenesis of leaves and flowers. This effect is often achieved by regulating the division and differentiation of cells.

In the course of research on cotton fiber development, the inventors cloned a gene GhTCP4 encoding a TCP-like protein that is specifically expressed in the elongation phase of fibroblasts. In order to further study the biological function of GhTCP4, the inventors performed cotton transgenic function analysis, constructed GhTCP4 overexpression and RNA interference (RNAi) vectors for cotton transformation, and successfully obtained multiple transgenic lines of each vector. Analysis of transgenic plants grown in greenhouses and fields showed that overexpression of GhTCP4 in cotton inhibited the elongation of fibroblasts, while inhibition of GhTCP4 expression promoted the elongation of fibroblasts, indicating that GhTCP4 plays a key negative role in cotton fiber elongation. Regulation. Gene expression assay showed that GhTCP4 also regulated the expression of GhRX1 and GhEXPA1 downstream genes regulated by GhHOX3. Protein interaction experiments demonstrated a direct protein interaction between GhTCP4 and GhHOX3. Further experiments demonstrated that the protein interaction of GhTCP4 with GhHOX3 attenuated the activation of GhHOX3 itself and the heterodimer formed by GhHOX3 and GhHD1 on the downstream genes GhRDL1 and GhEXPA1 promoter. These results indicate that the transcription factor GhTCP4 is an important regulator of cotton fiber cell development and has great potential in promoting cotton fiber elongation and fiber quality improvement. Use value.

The inventors conducted the following studies on the cotton GhTCP4 gene:

The complete GhTCP4 gene of two subgroups A and D was obtained from the upland cotton cultivar R15 by Genome walking and RACE technology.

2. Construction of GhTCP4 sense and double-stranded RNA interference transgenic vector: RDL1::mGhTCP4, 35S::dsGhTCP4.

3. Transgenic cotton was obtained by plant transformation using plant transgenic technology.

4. The 35S::dsGhTCP4 transgenic cotton obtained has a significantly increased trait of fiber length; the transgenic cotton of RDL1::GhTCP4 has traits with significantly shorter fiber length; these traits can be stably inherited.

Through the gene expression and protein interaction experiments, the mechanism of GhTCP4 regulating cotton fiber cell development was elucidated.

The invention discloses the GhTCP4 gene information and use for the first time, and it is very surprising to find that adjusting the expression amount can regulate the growth of cotton fiber cells, especially inhibiting the expression of GhTCP4 to promote the growth of fiber cells and increasing the length of cotton fibers, which is important for the quality improvement of cotton fibers. Value.

The GhTCP4 gene of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA or synthetic DNA. The genomic DNA may be the same as the sequence shown in SEQ ID NO.: 3 or a degenerate variant. The DNA of the present invention may be single-stranded or double-stranded, and the DNA may be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO.: 1 or a degenerate variant.

As used herein, a "degenerate variant" in the present invention refers to a protein having SEQ ID NO.: 2, but to the coding region sequence shown in SEQ ID NO.: 1 or SEQ ID NO.: The indicated genomic sequences differ in nucleic acid sequences.

Polynucleotides encoding the mature polypeptide of SEQ ID NO.: 2 include: coding sequences encoding only mature polypeptides; coding sequences for mature polypeptides and various additional coding sequences; coding sequences for mature polypeptides (and optionally additional coding sequences) And non-coding sequences.

The term "polynucleotide encoding a polypeptide" can be a polynucleotide comprising the polypeptide, or a polynucleotide further comprising additional coding and/or non-coding sequences.

The invention also relates to variants of the above polynucleotides which encode fragments, analogs and derivatives of polypeptides or polypeptides having the same amino acid sequence as the invention. Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide encoded thereby. .

The invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The invention particularly relates to polynucleotides that hybridize to the polynucleotides of the invention under stringent conditions. In the present invention, "stringent conditions" means: (1) hybridization and elution at a lower ionic strength and a higher temperature, such as 0.2 x SSC, 0.1% SDS, 60 ° C; or (2) Hybridization with a denaturing agent such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 °C, etc.; or (3) at least 90% identity between the two sequences Above, it is better that the hybridization occurs more than 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO.: 2.

The invention also relates to nucleic acid fragments that hybridize to the sequences described above. As used herein, a "nucleic acid fragment" is at least 15 nucleotides in length, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more. Nucleic acid fragments can be used in nucleic acid amplification techniques (such as PCR) to identify and/or isolate polynucleotides encoding cotton fiber/plant cell length related polypeptides.

Peptide encoded by GhTCP4 gene

As used herein, the terms "GhTCP4 polypeptide", "GhTCP4 protein", "cotton fiber length-related polypeptide", "polypeptide of the invention", "polypeptide encoded by the GhTCP4 gene" are used interchangeably and refer to the control cotton of the present invention. Fiber/plant cell length polypeptide.

In a preferred embodiment, the polypeptide of the invention is derived from cotton.

The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide. The polypeptides of the invention may be naturally purified products, either chemically synthesized or produced recombinantly from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plants, insects, and mammalian cells). The polypeptide of the invention may be glycosylated or may be non-glycosylated, depending on the host used in the recombinant production protocol. Polypeptides of the invention may also or may not include an initial methionine residue.

The invention also includes fragments, derivatives and analogs of the GhTCP4 polypeptide. As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that substantially retains the same biological function or activity of a native GhTCP4 polypeptide of the invention. The polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide and another compound (such as a compound that extends the half-life of the polypeptide, for example Polyethylene glycol) a polypeptide formed by fusion, or (iv) a polypeptide formed by fused an additional amino acid sequence to the polypeptide sequence (such as a leader or secretion sequence or a sequence or proprotein sequence used to purify the polypeptide, or fusion) protein). These fragments, derivatives and analogs are within the purview of those skilled in the art in light of the teachings herein.

In a preferred embodiment, the polypeptide of the invention refers to a polypeptide having the sequence of SEQ ID NO.: 2 which modulates the function of cotton fiber/plant cell length. Also included are variant forms of the sequence of SEQ ID NO.: 2 that have the same function as the GhTCP4 polypeptide. These variants include, but are not limited to, one or more (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10) amino acid deletions , Insertion and/or Substitution, and the addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, when substituted with amino acids of similar or similar properties, the function of the protein is generally not altered. As another example, the addition of one or several amino acids at the C-terminus and/or N-terminus will generally not alter the function of the protein. The term also encompasses active fragments and active derivatives of GhTCP4 polypeptides.

Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutations A variant, an inducible mutant, a protein encoded by DNA capable of hybridizing to the DNA of the GhTCP4 polypeptide under high or low stringency conditions, and a polypeptide or protein obtained using an antiserum against the GhTCP4 polypeptide. The invention also provides other polypeptides, such as fusion proteins comprising a GhTCP4 polypeptide or a fragment thereof. In addition to the nearly full length polypeptide, the invention also includes soluble fragments of the GhTCP4 polypeptide. Typically, the fragment has at least about 10 contiguous amino acids of the GhTCP4 polypeptide sequence, typically at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100. A contiguous amino acid.

The invention also provides a GhTCP4 polypeptide or analog thereof. The difference between these analogs and the native GhTCP4 polypeptide may be a difference in amino acid sequence, a difference in the modification form which does not affect the sequence, or a combination thereof. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by a variety of techniques, such as random mutagenesis by irradiation or exposure to a mutagen, or by site-directed mutagenesis or other techniques known to molecular biology. Analogs also include analogs having residues other than the native L-amino acid (such as D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (such as beta, gamma-amino acids). It is to be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.

Modifications (usually without altering the primary structure) include chemically derived forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation. Modified forms also include sequences having phosphorylated amino acid residues such as phosphotyrosine, phosphoserine, phosphothreonine. Also included are polypeptides modified to increase their resistance to proteolytic properties or to optimize solubility properties.

In the present invention, the "GhTCP4 polypeptide conservative variant polypeptide" means up to 10, preferably up to 8, more preferably up to 5, most preferably up to the amino acid sequence of SEQ ID NO.: 2. The three amino acids are replaced by amino acids of similar or similar nature to form a polypeptide. In the protein, when substituted with amino acids of similar or similar properties, the function of the protein is usually not changed, and the addition of one or several amino acids at the C-terminus and/or the end does not usually change the function of the protein. These conservative variant polypeptides are preferably produced by amino acid substitutions according to the following table.

cotton

Cotton is a seed fiber of the Govsaceae family of Malvaceae, native to the subtropical zone. The plants are shrubby and can grow up to 6 meters in the tropics, usually 1 to 2 meters. The flowers are milky white, and soon turn into a deep red after flowering and then wither, leaving a small green capsule called a cotton boll. There are cottonseed in the cotton boll, and the hair on the cottonseed grows from the cottonseed epidermis and is filled with the inside of the cotton boll. The cotton boll splits when it matures, revealing soft cotton fibers. Common cotton fibers are white to white with yellow, about 2 to 4 cm long, and contain about 87 to 90% of cellulose.

Landscaping (Gossypium hirsutum L.) is named after the earliest planting in the American continent. It is the most important cotton cultivar in the world, accounting for more than 90% of the global cotton planting area. Upland cotton is a heterotetraploid, including two subgenomics, subgroup A and subgroup D.

Recombination technology and plant improvement

The full-length sequence of the gene of the present invention or a fragment thereof can usually be obtained by a PCR amplification method, a recombinant method or a synthetic method. For PCR amplification, primers can be designed in accordance with the disclosed nucleotide sequences, particularly open reading frame sequences, and can be prepared using commercially available cDNA libraries or conventional methods known to those skilled in the art. The library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order.

Once the relevant sequences are obtained, the recombinant sequence can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.

In addition, synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short. Usually, a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then performing the ligation.

At present, it has been possible to obtain a DNA sequence encoding the protein of the present invention (or a fragment thereof, or a derivative thereof) completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

The invention also relates to vectors comprising the polynucleotides of the invention, as well as host cells genetically engineered using the vectors of the invention or the polypeptide coding sequences of the invention, and methods of producing the polypeptides of the invention by recombinant techniques.

The polynucleotide sequences of the present invention can be used to express or produce recombinant polypeptides of the present invention by conventional recombinant DNA techniques (Science, 1984; 224: 1431). Generally there are the following steps:

(1) transforming or transducing a suitable host cell with a polynucleotide (or variant) of the invention, or with a recombinant expression vector containing the polynucleotide;

(2) a host cell cultured in a suitable medium;

(3) Separating and purifying the protein from the culture medium or the cells.

The polynucleotide sequence of the present invention can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus or other vector well known in the art. In summary, any plasmid and vector can be used as long as it can replicate and stabilize in the host. An important feature of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and a translational control element.

Methods well known to those skilled in the art can be used to construct expression vectors containing the polynucleotides of the invention and suitable transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.

Vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences, can be used to transform appropriate host cells to enable expression of the protein.

The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell (such as a cell of a crop or a forestry plant). Representative examples are: Escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast; plant cells, and the like.

When a polynucleotide of the present invention is expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. An enhancer is a cis-acting factor of DNA, usually about 10 to 300 base pairs, acting on a promoter to enhance transcription of the gene.

It will be apparent to one of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells.

Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E. coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl ₂ method, and the procedures used are well known in the art. Another method is to use MgCl ₂ . Conversion can also be carried out by electroporation if desired. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.

Transformed plants can also be subjected to methods such as Agrobacterium transformation or gene gun transformation, such as the leaf disc method. For transformed plant cells, tissues or organs, plants can be regenerated by conventional methods to obtain plants having a change in cotton fiber length traits.

The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The cultivation is carried out under conditions suitable for the growth of the host cell. When the host cells grow to the appropriate cell density, use the appropriate The method (such as temperature conversion or chemical induction) induces the selected promoter and the cells are cultured for a further period of time.

The recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell. If desired, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, ultrafiltration treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption Chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

The recombinant polypeptide of the invention has a variety of uses. For example, for screening compounds, polypeptides or other ligands having a regulated cotton fiber/plant cell length. A library of screened polypeptides with expressed recombinant polypeptides of the invention can be used to find valuable polypeptide molecules that inhibit, or promote, elongation of cotton fibers/plant cells.

In another aspect, the invention also encompasses polyclonal and monoclonal antibodies, particularly monoclonal antibodies, that are specific for the polypeptides of the invention. The present invention encompasses not only intact monoclonal or polyclonal antibodies, but also immunologically active antibody fragments, or chimeric antibodies.

Antibodies of the invention can be prepared by a variety of techniques known to those skilled in the art. For example, a purified gene product of a polypeptide of the present invention or a fragment thereof having antigenicity can be administered to an animal to induce production of a polyclonal antibody. The various antibodies of the invention can be obtained by conventional immunological techniques using fragments or functional regions of cotton fiber length related gene products. These fragments or functional regions can be prepared by recombinant methods or synthesized using a polypeptide synthesizer. An antibody that binds to an unmodified form of a cotton fiber length-related gene product can be produced by immunizing an animal with a gene product produced in a prokaryotic cell (eg, E. coli); an antibody that binds to a post-translationally modified form (eg, glycosylation or phosphoric acid) The protein or polypeptide can be obtained by immunizing an animal with a gene product produced in a eukaryotic cell such as yeast or insect cells. Antibodies against the polypeptides of the invention can be used to detect cotton fiber length related polypeptides in a sample.

The invention also relates to a test method for quantifying and localizing the level of cotton fiber length related polypeptides. These tests are well known in the art. The cotton fiber length-related polypeptide levels detected in the test can be used to explain the function of cotton fiber length-related polypeptides to regulate cotton fiber length.

A method for detecting the presence or absence of a cotton fiber length-related polypeptide in a sample is carried out by using a specific antibody of the polypeptide of the present invention, which comprises: contacting a sample with a specific antibody of the polypeptide of the present invention; observing whether an antibody complex is formed, forming an antibody The complex means that a cotton fiber length related polypeptide is present in the sample.

A part or all of the polynucleotide of the present invention can be immobilized as a probe on a microarray or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis of genes in tissues. Transcription products of the polypeptides of the invention can also be detected by RNA-polymerase chain reaction (RT-PCR) in vitro amplification using primers specific for the polypeptides of the invention.

The main advantages of the invention include:

(a) provides a GhTCP4 gene capable of regulating the length of cotton fiber/plant cells;

(b) inhibiting or promoting the elongation of cotton fibers by up-regulating or down-regulating the expression level of GhTCP4;

(c) The degree of down-regulation of GhTCP4 expression is proportional to the extent of fiber growth.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually in accordance with conventional conditions or according to the conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

Example 1

GhTCP4 full-length gene clone

1. Cotton RNA extraction

Cotton RNA was extracted using the cold phenol method. Collecting upland cotton R15 (land cotton variety “Jinmian R15”, purchased from the Cotton Research Institute of Shanxi Academy of Agricultural Sciences). The fiber on the surface of the ovule 6 days after flowering was ground into powder in liquid nitrogen and transferred to a 50 mL centrifuge tube. Add 8 mL of extraction buffer (1 M Tris-HCl, 50 mM EDTA, 1% SDS, pH 9.0) and an equal volume of water-saturated phenol: chloroform: isoamyl alcohol (25:24:1), shake and mix, place on ice 1h, mix once every 10 minutes. Centrifuge at 13,000 g for 20 min at 4 °C. Repeat phenol: chloroform: isoamyl alcohol extraction 2 to 4 times, and finally extracted with chloroform: isoamyl alcohol (24:1). On the night, 1/2 volume of high salt solution (0.8 M sodium citrate, 1.2 M NaCl) and 1/2 volume of isopropanol were added, mixed, and placed at -70 ° C for 1 h. After centrifugation at 13,000 g for 20 min at 4 ° C, the supernatant was removed, and the precipitate was dissolved in 1 mL of DEPC-treated water, and centrifuged at 13,000 g for 10 min at 4 ° C. The supernatant was transferred to a 1.5 mL Eppendorf tube, 1/3 volume of 8 M LiCl and volume of NaAC was added and left at -20 °C overnight. Centrifuge at 13,000 g for 20 min at 4 °C. The supernatant was removed, and the precipitate was washed twice with 1 mL of 70% ethanol, and blown at room temperature for 20 min, and dissolved in 100-200 μL of DEPC-treated water.

2. According to the sequence of GhTCP4 cDNA, synthesize specific primers (including restriction sites and protective bases):

The reverse transcription product of total RNA from 6 days after flowering was used as template to carry out PCR reaction. The reaction conditions were pre-denaturation at 94 °C for 5 min; then denaturation at 94 °C for 30 s, 56 °C for 30 s, and 72 ° C for 1 min for 35 cycles. Finally extended at 72 ° C for 10 min. The PCR product was purified by electrophoresis, recovered and subcloned into the commercial vector pMD18-T, and sequenced to confirm the sequence was correct.

Combined with Genome walking and RACE technology, the complete GhTCP4 genes of A and D subgroups were obtained from the heterologous tetraploid upland cotton (Gossypium hirsutum) cultivar R15, named GhTCP4A and GhTCP4D. A similar approach was used to amplify the TCP4 gene sequence from other cotton varieties.

The results showed that GhTCP4 encodes a TCP protein of 401 amino acids with a TCP domain that is highly similar to the Arabidopsis TCP4 conserved domain (Fig. 1A, B). The similarity of GhTCP4A and GhTCP4D at protein level is as high as 97%, and the homology of TCP4 protein in different cotton species is also between 96.5% and 100% (Fig. 1C). It is speculated that the TCP4 protein functions of two subgroups of upland cotton are basically the same. Pyrosequencing analysis showed that GhTCP4A and GhTCP4D were simultaneously expressed during cotton fiber development (Fig. 2B).

Example 2

Construction of 35S::dsGhTCP4 and RDL1::mGhTCP4 Expression Vectors and Agrobacterium Transformation

1. Construction of 35S::dsGhTCP4 plant expression vector

The high-fidelity enzyme amplifies the selected GhTCP4 RNA interference (RNAi) fragment, the forward fragment introduces the SmaI and XbaI restriction sites, the reverse fragment introduces the SacI and NotI restriction sites, and the forward and reverse sequences are separately cloned into the Both ends of the RTM sequence on the PBSK vector of the RTM intron were digested with SmaI and SacI to replace the GUS gene on 35S::NOS/p121 to form a 35S::dsGhTCP4 plant expression vector, and the sequence was verified to be correct by sequencing.

The GhTCP4 RNA interference sequence is set forth in SEQ ID NO.: 4.

Primer sequence for RNA interference:

2. Construction of RDL1::mGhTCP4 expression vector

(1) A promoter fragment of about 700 bp upstream of the coding region of the GhRDL1 gene, and a NOS terminator were cloned into the PCAMBIA-2301 vector HindIII, PstI and SacI, and EcoRI sites, respectively, to form an RDL1::2301 intermediate vector.

(2) Two rounds of PCR site-directed mutagenesis of GhTCP4, the principles and steps are as follows:

In the first round of PCR, the fragments on both sides of the mutation site were amplified by high-fidelity enzyme, and the primers were P1-P3, P4-P2. The two PCR products were separately collected by tapping, and 0.1 μL of each was mixed as a second round PCR template, and the primers were P1-P2.

The site to which GhTCP4 was targeted by mi319 was mutated according to the method shown in FIG. After the mutant mGhTCP4 was confirmed by sequencing, it was amplified by high-fidelity PCR and introduced into a suitable restriction site, and cloned into the expression vector RDL1::NOS/2301 to form RDL1::mGhTCP4 plant expression vector, and the sequence was verified by sequencing. (See Example 1).

Site-directed mutagenesis primer sequence:

3. Agrobacterium tumefaciens transformation

The transformation of Agrobacterium tumefaciens is carried out by freeze-thaw method. One single colony LBA4404 or GV3101 (Invitrogen), 3 mL LB medium (25 μg/mL rifamycin Rif and 50 μg/mL kanamycin Kan or gentamicin Gen) was incubated at 28 ° C, 220 rpm overnight. 2 mL of bacterial solution, 50 mL of LB medium (25 μg/mL Rif and 50 μg/mL Gen), cultured at 28 ° C, 220 rpm, to OD ₆₀₀ = 0.5 (about 6 h). Place on ice for 30 min, 4 ° C, and centrifuge at 5000 g for 5 min. Resuspend in 10 mL of 0.15 M NaCl. Centrifuge at 5000 g for 5 min at 4 °C. Resuspend in 1 mL of 20 mM CaCl ₂ , dispense at 50 μL/tube, freeze at liquid nitrogen, and store competent cells at -70 °C. The target gene binary vector and 50 μL/tube competent cells were mixed, placed on ice for 30 minutes, and frozen in liquid nitrogen for 1 min. The bacterial solution was thawed in a 37 ° C water bath for 5 min, and 1 mL of LB medium was added, and the mixture was cultured at 28 ° C, 220 rpm for 2 to 4 hours. 50-100 μL of LB medium plate (25 μg/mL Rif, 50 μg/mL Gen and 50 μg/mL kanamycin Kan or hygromycin Hyg) was taken. After 2 days, single colonies were picked for PCR identification.

Example 3

Plant transformation and screening of transgenic offspring

Agrobacterium containing RDL1::mGhTCP4 and 35S::dsGhTCP4 vector plasmids were cultured on YEB bacterial medium supplemented with kanamycin 50 mg/L, rifampicin 100 mg/L and streptomycin 300 mg/L for 2 to 3 days, respectively. The single colonies were inoculated into YEB liquid medium containing the same antibiotic, and cultured in suspension at 28 ° C, 200 rpm / min on a shaker overnight. The bacterial solution was centrifuged at 4000 rpm/min for 10 min, and the pellet was resuspended in 1/2 MS liquid medium containing 30 g/L of glucose and 100 μmol/L of acetosyringone, and the OD ₆₀₀ value was adjusted to about 0.4 to 0.6, which was used as an infection solution.

Cotton R15 seed (Jinnian R15 variety) was routinely disinfected and placed in 1/2MS0 (1/2MS salt + 5g / L glucose + 7g / L agar powder, pH 6.0) medium, germinated in the dark, 5 ~ 7 After the day, the sterile hypocotyls were cut into segments of about 1.0 cm as replacement for explants.

The explants were immersed in Agrobacterium liquid for 15-20 min and transferred to co-culture medium MSB1 (MS salt + B5 organic + 30 g / L glucose + 0.1 mg / L KT + 0.1 mg / L 2, 4-D + 2.2g/L Gelrite, pH 6.0), after dark culture at 22 °C for 2 days, the explants were transferred to medium MSB2 (MSB1 + 500mg / L cephalosporin + 80mg / L kanamycin) for callus Induction. Explants were induced by resistant callus, callus proliferation and embryogenic callus induction (medium MSB3: MS salt + B5 organic + 30 g / L glucose + 2.5 g / L Gelrite, pH 6.0), Somatic embryogenesis (medium MSB4: MS salt + B5 organic + 30 g / L glucose + 1.0 g / L asparagine + 2.0 g / L glutamine + 3.0 g / L Gelrite, pH 6.0; MS salt KNO _{3 is} doubled, NH ₄ NO ₃ ) is removed, and resistant test tube seedlings are regenerated. When the test tube seedlings grow to 3-4 true leaves, they are transplanted into pots and placed in an artificial climate chamber for growth.

The results are shown in Figure 3. Overexpression of anti-miR319 cleavage of GhTCP4 in cotton resulted in leaf phenotypic changes (Fig. 3A-B). Subcellular localization revealed that GhTCP4-GFP protein localizes to the nucleus of tobacco leaf epidermal cells (Fig. 3C). ), suggesting that GhTCP4 plays a transcriptional regulatory function in fibroblasts.

Example 4

Molecular biological identification of transgenic plants

PCR

DNA extraction was performed using a cold phenol method. 2 g of material, ground into powder in liquid nitrogen, transferred to a 50 mL centrifuge tube, 8 mL of extraction buffer (1 M Tris-HCl, 50 mM EDTA, 1% SDS, pH 9.0) and an equal volume of water-saturated phenol: chloroform: Isoamyl alcohol (25:24:1), shake and mix, place on ice for 1 h, mix once every 10 min. Centrifuge at 13,000 g for 20 min at 4 °C. Repeat phenol: chloroform: isoamyl alcohol extraction 2 to 4 times, and finally extracted with chloroform: isoamyl alcohol (24:1). Take the night and add 1/2 volume of high salt The solution (0.8 M sodium citrate, 1.2 M NaCl) and 1/2 volume of isopropanol were mixed and placed at -70 ° C for 1 h. After centrifugation at 13,000 g for 20 min at 4 ° C, the supernatant was removed, and the pellet was washed twice with 1 mL of 70% ethanol, and sprinkled for 20 min at room temperature, and the precipitate was dissolved in 1 mL of sterile water. Centrifuge at 13000 g for 10 min at 4 °C. The supernatant was taken, 5-10 μL of RNase (10 mg/mL) was added, and digestion was carried out at 37 ° C for 30 min.

a. PCR identification of transgenic positive cotton using NPT II specific primers:

The reaction conditions were: pre-denaturation at 94 ° C for 5 min; then pre-denaturation at 94 ° C for 30 sec, reflux at 56 ° C for 30 sec, extension at 72 ° C for 1 min, for a total of 35 cycles; and finally at 72 ° C for 10 min. The amplified fragment size is approximately 680 bp.

b.35S::dsGhTCP4 transgenic cotton was identified using transgenic vector-specific primers:

The reaction conditions were: pre-denaturation at 94 ° C for 5 min; then pre-denaturation at 94 ° C for 30 sec, reflux at 56 ° C for 30 sec, extension at 72 ° C for 25 sec for 35 cycles, and finally at 72 ° C for 10 min. The amplified fragment size is approximately 280 bp.

c. Identification of RDL1::mGhTCP4 transgenic cotton using transgenic vector-specific primers:

The reaction conditions were: pre-denaturation at 94 ° C for 5 min; then pre-denaturation at 94 ° C for 30 sec, reflux at 56 ° C for 30 sec, extension at 72 ° C for 30 sec, for a total of 35 cycles; and finally at 72 ° C for 10 min. The amplified fragment size is approximately 340 bp.

2. Fluorescence quantitative RT-PCR analysis

a.RT-PCR analysis

1 μg of total RNA, reverse transcription by Oligo (dT) primer, 20 μL of reaction solution. The reaction was carried out at 42 ° C for 30 minutes, and the first strand cDNA was reverse transcribed and left overnight. 0.5 μL of the reverse transcription product was taken, and 25 μL of the PCR reaction system was used to detect the expression of the target gene. The PCR primers are specific primers for GhTCP4:

The amount of template for the RT-PCR reaction was corrected using the cotton gene Histone 3 (AF024716) as an internal standard. The PCR reaction conditions were: predenaturation at 94 ° C for 5 minutes; then denaturation at 94 ° C for 30 seconds, renaturation at 56 ° C for 30 seconds, extension at 72 ° C for 1 minute for a total of 30 cycles; and finally 72 ° C for 10 minutes.

b. Fluorescence quantitative RT-PCR analysis

Quantitative RT-PCR detection using chimeric fluorescence

Premix Ex Taq ^TM II (Perfect Real Time), TaKaRa, DRR041A). The reaction system is as follows:

The cotton gene Histone 3 (AF024716) was used as an internal standard. Data analysis was performed using Realplex v2.0 (available from Eppendorf, NSW, Germany). The experiment was repeated three times, and the average and variance of each group of data were taken to draw a chart.

Expression analysis showed that the expression of GhTCP4 gradually increased after the differentiation of cotton fiber cells, and maintained a high level in the elongation phase (6-12DPA) fiber, and then gradually decreased (Fig. 2A). In the tetraploid island cotton, the homologous genes of GhTCP4 in cotton fiber cells of diploid Asian cotton also showed similar expression characteristics (Fig. 2C, D). The cotton TCP4 gene is highly expressed in rapidly elongating fibroblasts and may function during elongational growth of fibroblasts.

Example 5

Analysis of fiber length and gene expression of transgenic cotton

1. Analysis of traits of transgenic cotton

35S::dsGhTCP4, RDL1::mGhTCP4 transgenic cotton and wild-type R15 were planted in Shanghai and Hainan farms, and were routinely managed in the field. During the mature boll opening period of cotton, mature cotton peaches were collected from each plant, and the consistency of the collected parts was kept as much as possible. A certain number of seeds were randomly taken to flatten the fibers and measure the fiber length for statistical analysis. The results showed that the phenotype of 35S::dsGhTCP4 transgenic cotton could be stably inherited and showed a trend of increasing from generation to generation, which may be related to the homozygous insertion of transgene insertion.

Statistics on mature cotton fibers showed that the cotton fiber length of the transgenic cotton was significantly increased compared with the control, and the more the GhTCP4 expression was down-regulated, the more the fiber growth was more obvious (Fig. 4A-C). This negative correlation indicates that the high expression of GhTCP4 in cotton fiber cells has an inhibitory effect on its elongation growth, and inhibition of GhTCP4 activity can promote the elongation of fiber cells.

Gene expression analysis of RDL1::mGhTCP4 transgenic cotton showed that the expression of GhTCP4 in RDL1::mGhTCP4 fibroblasts was much higher than that in the control group of the same period (Fig. 5A). T2 generation RDL1::mGhTCP4 cotton showed a significantly shorter phenotype (Fig. 5B, C). The growth of epidermal hairs in other parts of the plant, such as leaves and stem nodes, was also significantly inhibited, indicating GhTCP4 activity. The enhancement inhibits the elongation of cotton fibers.

Example 6

Molecular mechanism of GhTCP4 regulating cotton fiber development

It was found by yeast two-hybrid assay that GhTCP4 interacts with GhHOX3, and GhHOX3 is segmented according to different domains and analyzed by yeast two-hybrid assay. The results indicate that the LZ+START domain of GhHOX3 mediates the protein interaction of GhHOX3 with GhTCP4 (Fig. 6A). The LZ domain has been reported to be required for homebox-like protein binding to DNA.

Semi-molecule fluorescence complementation (BiFC) experiments based on firefly luciferase (LUC) showed that GhTCP4 and GhHOX3 form stable protein complexes in tobacco cells (Fig. 6B). Further protein immunoprecipitation (CoIP) experiments also confirmed the direct protein interaction between GhTCP4 and GhHOX3 in plants (Fig. 6C).

Studies have shown that GhHOX3 activates gene expression by directly binding to the L1-box of the GhRDL1 and GhEXPA1 promoter regions. In order to analyze the physiological significance of interaction between GhTCP4 and GhHOX3, a dual fluorescence reporter system of LUC gene driven by GhRDL1 and GhEXPA1 promoters (with NR as the internal reference) was constructed, and GhTCP4 and GhHOX3 were used as effector. The response of sub-activity to different protein combinations (Figure 7).

The results showed that the transient expression of GhHOX3 strongly enhanced the GhRDL1 and GhEXPA1 promoter activities compared with the control, while the expression of GhTCP4 alone did not affect the activities of GhRDL1 and GhEXPA1 promoters; when co-expressing GhHOX3 and GhTCP4, GhHOX3 to GhRDL1 and GhEXPA1 The activation effect of the promoter was significantly inhibited (Fig. 7A, B). The above results indicate that GhTCP4 inhibits downstream gene expression through interaction with GhHOX3.

The formation of heterodimers between GhHOX3 and another homeobox-like protein GhHD1 is more active for transcriptional activation of downstream genes. Activation of the GhRDL1 or GhEXPA1 promoter by the GhHD1-GhHOX3 heterodimer was also found to be strongly inhibited by GhTCP4 (Fig. 7C, D). Combining the expression data of genes in transgenic cotton fiber cells, the above results demonstrate that the interaction of GhTCP4 with GhHOX3 inhibits the activation of downstream genes by GhHOX3, thereby regulating the elongation and growth of fibroblasts.

All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims (10)

1. Use of a GhTCP4 gene or a protein encoded thereby, wherein the GhTCP4 gene or the encoded protein thereof is used for one or more uses selected from the group consisting of:

   (a) for the preparation of an agent or composition that promotes elongation of a plant cell;

   (b) for promoting elongation of plant cells;

   (c) for the preparation of a reagent or composition for promoting elongation of cotton fibers;

   (d) Used to promote elongation of cotton fibers.
2. The use according to claim 1, wherein said plant cells are cotton fiber cells.
3. The use according to claim 1, wherein the CDS sequence of the GhTCP4 gene is as shown in SEQ ID NO.: 1.
4. A method for regulating the length of a plant cell, comprising the steps of:

   (a) introducing a foreign construct into a plant cell, wherein the construct contains an exogenous GhTCP4 gene sequence, an exogenous nucleotide sequence that promotes expression of the GhTCP4 gene, or an exogenous nucleotide that inhibits expression of the GhTCP4 gene. a sequence to obtain a plant cell into which the exogenous construct is introduced;

   (b) the plant cells introduced into the exogenous construct obtained in the previous step are regenerated into plants: and

   (c) optionally identifying the regenerated plant to obtain a plant having a change in plant cell length.
5. A method for promoting elongation of cotton fibers, characterized in that the method comprises the steps of inhibiting the expression of the GhTCP4 gene or inhibiting the activity of the GhTCP4 protein in the plant.
6. The method of claim 5 wherein said promoting cotton fiber elongation comprises promoting elongation of cotton fiber cells.
7. The method of claim 5, wherein the method comprises introducing into the plant an exogenous nucleotide sequence that inhibits expression of the GhTCP4 gene.
8. The method of claim 7, wherein said exogenous nucleotide sequence comprises an RNAi interference sequence.
9. The method of claim 5 wherein said method comprises the steps of:

   (a) Agrobacterium providing an expression vector carrying the GiTCP4 gene RNAi interference sequence;

   (b) contacting the plant cell or tissue or organ with the Agrobacterium in step (a), thereby transferring the GhTCP4 gene RNAi interference sequence into the plant cell and integrating it into the chromosome of the plant cell;

   (c) selecting a plant cell or tissue or organ that has been transferred into the GhTCP4 gene RNAi interference sequence;

   (d) regenerating the plant cell or tissue or organ in step (c) into a plant.
10. Use of a modulator of the GhTCP4 gene or a protein encoded thereby, for use in one or more uses selected from the group consisting of:

    (a) an agent or composition for preparing a plant cell length;

    (b) for regulating the length of plant cells;

    (c) an agent or composition for preparing a cotton fiber length; and

    (d) used to regulate the length of cotton fibers.

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