CN121718556A - Function of cotton receptor-like kinase GhTMK1 and application thereof in improving cotton fiber quality - Google Patents

Abstract

The invention relates to a receptor-like kinase protein GhTMK which participates in plant growth and development regulation and application thereof in cotton. Specifically, ghTMK genes are separated and cloned from cotton, the expression quantity of the cotton GhTMK genes is regulated and controlled through molecular biology and transgenic technology, the regulation of plant growth and development is realized, and the transgenic cotton with obviously increased mature fiber length is obtained. The invention discloses functions and applications of cotton receptor-like kinase protein GhTMK, which have positive effects in promoting the elongation of fiber cells and improving the length quality characteristics of cotton fibers, and have wide application prospects.

Description

Function of cotton receptor-like kinase GhTMK1 and application thereof in improving cotton fiber quality

Technical Field

The invention relates to the fields of biotechnology and plant biology, in particular to cotton receptor-like kinase protein GhTMK1 and application thereof.

Background

Cotton is an important commercial crop, providing a large number of natural textile materials for the market. Therefore, improving cotton fiber quality is an important issue for cotton breeding. The cotton fiber is the single cell surface coat on cotton seeds, and is formed by single cell development of cotton ovule epidermis, and the differentiation and development process can be divided into 4 periods of differentiation and protrusion of fiber primitive cells, rapid elongation of fiber cells, synthesis of secondary cell walls, dehydration and maturation and the like. Vital movements of the fibre cells at various stages affect the yield and quality formation of cotton fibres, wherein fibre elongation phase development affects both the yield and quality of cotton fibres.

Cotton fiber is one of the longest plant cells, and the length of upland cotton fiber is generally 25-30 mm, and the length of island cotton fiber is generally above 35 mm. The cotton fiber is polar and very rapid, the length of the mature fiber is 1000-3000 times of its diameter, and the cell elongation speed can be as high as 2 mm/day in the rapid elongation stage. Research on the regulation mechanism of cotton fiber elongation helps us to understand better the mechanism of polar elongation of plant cells.

The plant transmembrane kinase TMK family gene codes for LRR plant receptor-like kinase, and its protein consists of extracellular LRR domain, a transmembrane domain and intracellular kinase domain. TMK is used as an auxin signal component, and coordinates plant growth and development by regulating cell expansion and proliferation. On the cell membrane, TMK and auxin binding proteins ABP1/ABLs constitute receptor complexes that sense extracellular auxin and are activated by auxin, and the activated TMK directly phosphorylates a range of effector proteins including atypical AUX/IAA proteins IAA32/34, proton pump AHA, MKK4/5-MPK3/6, tryptophan transferase TAA1, ABI1/2, and the like, thereby regulating rapid auxin response and various plant development processes. On the day of cotton flowering, the accumulation of auxin in the fibroblasts is highest, causing the fibroblasts to initiate and rapidly elongate. However, it is not known whether TMK, which senses auxin signals and mediates auxin rapid responses, is involved in regulating fibroblast development.

Disclosure of Invention

The invention provides an application of GhTMK gene in improving length quality character of plant fiber. The present invention provides a nucleic acid construct comprising

(1) Nucleic acid sequence encoding GhTMK protein, or

(2) Nucleic acid sequences which specifically interfere with the transcription and/or expression of GhTMK genes.

In one or more embodiments, the GhTMK1 protein has an amino acid sequence as set forth in SEQ ID NO. 1 or 2 or a sequence having at least 80% identity thereto.

In one or more embodiments, the nucleic acid sequence encoding GhTMK protein is shown as SEQ ID NO 3 or 4 or a sequence having at least 80% identity thereto.

In one or more embodiments, the GhTMK1 gene has NCBI numbers Gh_A13G0257 and/or Gh_D13G0274.

In one or more embodiments, the nucleic acid sequence of (2) is as set forth in SEQ ID NO. 5 or a sequence having at least 80% identity thereto.

In one or more embodiments, the nucleic acid construct further comprises a promoter operably linked to the nucleic acid sequence. Preferably, the promoter is a 35S promoter.

In one or more embodiments, the nucleic acid construct has elements from 5 'to 3' that are a promoter, a nucleic acid sequence encoding GhTMK a protein, and a terminator.

The present invention provides a genetically engineered host cell which

(1) Expression, inclusion or secretion of GhTMK proteins, or

(2) Comprising a nucleic acid construct as described in any of the embodiments herein.

In one or more embodiments, the host cell is not a plant cell.

The invention provides a method for regulating plant fiber cell elongation or regulating plant fiber length, which comprises the steps of up-regulating GhTMK protein expression or activity in a plant so as to promote plant fiber cell elongation or up-regulating plant fiber length, and down-regulating GhTMK protein expression or activity in the plant so as to inhibit plant fiber cell elongation growth or down-regulating plant fiber length.

In one or more embodiments, the plant is a malvaceae plant.

In one or more embodiments, the plant is a cotton plant.

In one or more embodiments, the plant is upland cotton (Gossypium hirsutum).

In one or more embodiments, the up-regulating expression in a GhTMK protein in a plant comprises transferring the coding sequence of the GhTMK1 protein into a plant to obtain a transformed plant.

In one or more embodiments, the down-regulating expression in a GhTMK protein in a plant comprises:

(1) Specific interference GhTMK with gene transcription and/or expression,

(2) Down-regulating GhTMK protein activity,

(3) GhTMK1 proteins with reduced activity are expressed in plants.

In one or more embodiments, the interference of (1) is interference with transcription of GhTMK genes or translation of their transcripts.

In one or more embodiments, (2) the down-regulating is included in a plant:

(i) Specific antibodies or ligands (e.g., inhibitory antibodies) expressing GhTMK1 protein capable of down-regulating GhTMK protein activity, or

(Ii) Introducing a nucleic acid sequence encoding (i) and/or a nucleic acid construct capable of expressing (i).

In one or more embodiments, the GhTMK1 protein has an amino acid sequence as set forth in SEQ ID NO. 1 or 2 or a sequence having at least 80% identity thereto.

In one or more embodiments, the nucleic acid sequence encoding GhTMK protein is shown as SEQ ID NO 3 or 4 or a sequence having at least 80% identity thereto.

In one or more embodiments, the GhTMK1 gene has NCBI numbers Gh_A13G0257 and/or Gh_D13G0274.

In one or more embodiments, the down-regulating expression in a GhTMK protein in a plant comprises transferring a nucleic acid that specifically interferes with transcription and/or expression of a GhTMK gene into a plant to obtain a transformed plant.

In one or more embodiments, the nucleic acid that specifically interferes with GhTMK with transcription and/or expression of the gene is selected from the group consisting of (1) an antisense nucleic acid, microRNA, siRNA, RNAi, dsRNA, sgRNA, or a combination thereof, and (2) a nucleic acid construct capable of expressing or forming (1).

In one or more embodiments, the nucleic acid that specifically interferes with the transcription and/or expression of the GhTMK gene is shown as SEQ ID NO 5 or a sequence having at least 80% identity thereto. In one or more embodiments, the sequence of the nucleic acid is operably linked to a promoter. Preferably, the promoter is a 35S promoter.

The invention provides an application of a substance for regulating GhTMK protein expression or activity in plants in regulating plant fiber cell elongation or regulating plant fiber length.

In one or more embodiments, the agent is an enhancer of GhTMK protein expression or activity, and the modulation is up-regulating GhTMK protein expression or activity in the plant, thereby promoting plant fiber cell elongation or up-regulating plant fiber length.

In one or more embodiments, the promoter is GhTMK protein or a coding sequence thereof.

In one or more embodiments, the agent is an inhibitor of GhTMK protein expression or activity, and the modulation is down-regulating GhTMK protein expression or activity in the plant, thereby inhibiting plant fiber cell elongation or down-regulating plant fiber length.

In one or more embodiments, the inhibitor is selected from the group consisting of:

(1) Inhibitors which specifically interfere with the transcription and/or expression of GhTMK gene,

(2) Inhibitors that down-regulate GhTMK protein activity,

(3) A GhTMK protein variant whose activity is down-regulated, or a coding sequence therefor.

In one or more embodiments, the inhibitor (1) is selected from the group consisting of (i) an antisense nucleic acid, microRNA, siRNA, shRNA, dsRNA, sgRNA, or a combination thereof, and (ii) a nucleic acid construct capable of expressing or forming (i).

In one or more embodiments, the inhibitor of (1) is a nucleic acid that specifically interferes with the transcription and/or expression of GhTMK gene, the sequence of which is shown as SEQ ID NO 5 or a sequence having at least 80% identity thereto.

In one or more embodiments, (2) the inhibitor is selected from the group consisting of (i) an antibody or ligand specific for GhTMK a protein, and (ii) a nucleic acid sequence encoding and/or a nucleic acid construct capable of expressing (i).

In one or more embodiments, the GhTMK1 protein has an amino acid sequence as set forth in SEQ ID NO. 1 or 2 or a sequence having at least 80% identity thereto.

In one or more embodiments, the nucleic acid sequence encoding GhTMK protein is shown as SEQ ID NO 3 or 4 or a sequence having at least 80% identity thereto.

In one or more embodiments, the GhTMK1 gene has NCBI numbers Gh_A13G0257 and/or Gh_D13G0274.

The invention also provides a method for screening plants for fiber length, which comprises comparing the expression or activity of plant GhTMK gene with that of wild type plant, if GhTMK gene expression or activity is up-regulated, plant fiber cell elongation growth is enhanced or plant fiber is longer, if GhTMK gene expression or activity is down-regulated, plant fiber cell elongation growth is inhibited or plant fiber is shorter.

In one or more embodiments, the GhTMK1 protein has an amino acid sequence as set forth in SEQ ID NO. 1 or 2 or a sequence having at least 80% identity thereto.

In one or more embodiments, the nucleic acid sequence encoding GhTMK protein is shown as SEQ ID NO 3 or 4 or a sequence having at least 80% identity thereto.

In one or more embodiments, the GhTMK1 gene has NCBI numbers Gh_A13G0257 and/or Gh_D13G0274.

The invention provides an application of GhTMK gene as a molecular marker for identifying the length of plant fiber.

In one or more embodiments, the use includes performing the steps of comparing the expression or activity of the plant GhTMK gene to a wild-type plant, if the expression or activity of the GhTMK gene is up-regulated, the plant fiber cell elongation growth is enhanced or the plant fiber is longer, and if the expression or activity of the GhTMK gene is down-regulated, the plant fiber cell elongation growth is inhibited or the plant fiber is shorter.

The present invention provides a method for obtaining a transgenic plant comprising the steps of:

(1) Providing an Agrobacterium harboring a nucleic acid construct as described in any of the embodiments herein,

(2) Contacting a cell or tissue or organ of a plant with the agrobacterium of step (1), thereby transferring the nucleic acid construct into the plant tissue or organ.

(3) Selecting a plant tissue, organ or seed into which has been transferred a nucleic acid sequence that is (a) a nucleic acid sequence encoding a GhTMK protein, or (b) a nucleic acid sequence that specifically interferes with transcription and/or expression of a GhTMK gene, and

(4) Regenerating the plant tissue, organ or seed of step (3).

Drawings

FIG. 1 is a upland cotton TMK protein family analysis.

(A) The phylogenetic tree of the upland cotton and Arabidopsis TMK protein family constructed by utilizing MEGA software adopts an adjacent method (Neighbor-Joining). The red color is labeled GhTMK1. (B) Upland cotton TMK protein family gene expression calorimetric diagram constructed according to the disclosed upland cotton transcriptome data. The red color is labeled GhTMK1.

FIG. 2 is a characterization of upland cotton GhTMK1 expression.

And detecting GhTMK1 expression quantities in different tissues and fibers in different development periods of upland cotton by qRT-PCR. The histone gene GhHIS of upland cotton (gh_d03d037070) was used as an internal reference gene (Shan et al, 2014). DPA (day post-anthesis) represents the days after flowering.

FIG. 3 is a 35S:: ghTMK1 transgenic cotton fiber phenotyping.

The method comprises the steps of (A) qRT-PCR detection of the expression quantity of GhTMK1 in the fiber 9 days after flowering, (B) 35S of GhTMK1 transgenic cotton fiber lengthening phenotype, and (C) 35S of GhTMK1 transgenic cotton fiber length statistics. Error bars are shown as standard deviation (STDEV); "indicates that the experimental and control groups were analyzed by the double sample equal variance hypothesis student's t test, P <0.0001, n=5.

FIG. 4 is a 35S:: dsGhTMK1 transgenic cotton fiber phenotyping.

The method comprises the steps of (A) qRT-PCR detection of the expression quantity of GhTMK1 in the fiber 9 days after flowering, (B) 35S of dsGhTMK1 transgenic cotton fiber shortening phenotype, and (C) 35S of dsGhTMK1 transgenic cotton fiber length statistics. Error bars are shown as standard deviation (STDEV) and the experimental and control groups were analyzed by the double-sample equal variance hypothesis student's t test, "+" indicates P <0.0001, n=5.

FIG. 5 is a GhTMK protein subcellular localization observation.

Fluorescent confocal microscopy observation of RDL shows that the subcellular localization of GhTMK1 protein in the fiber cells 1 day after flowering of GhTMK-YFP transgenic cotton shows that GhTMK protein is localized at the fiber cell membrane and significantly redundant other positions are localized at the top of the fiber cell membrane. Bright represents the Bright field channel, YFP represents the yellow fluorescent channel, merged represents the combination of the Bright field channel and the yellow fluorescent channel.

Detailed Description

The inventor firstly discloses that the plant fiber cell elongation can be obviously regulated or the plant fiber length can be regulated by directionally regulating and controlling GhTMK gene expression level in the plant, so that a new plant germplasm with improved fiber quality can be cultivated.

As used herein, "plant fiber" is a thick-walled tissue that is widely distributed in seed plants,

Its cells are elongated and sharp at both ends with thicker secondary cell walls. Preferably, the plant is a malvaceae plant, more preferably the plant is a cotton plant.

In a first aspect the present invention provides the use of a substance selected from the group consisting of GhTMK gene or its encoded protein, or its promoter or inhibitor, for regulating plant fibre cell elongation or regulating plant fibre length.

"GhTMK1 encoding protein", "GhTMK1 protein" or "GhTMK (not italic)" refers to a polypeptide having GhTMK1 activity, including but not limited to variant forms of the polypeptide. "variant forms" include, but are not limited to, deletions, insertions and/or substitutions of several (usually 1-50, preferably 1-20, 1-10, 1-8, 1-5) amino acids, and additions or deletions 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, substitution with amino acids of similar or similar properties does not generally alter the function of the protein. Amino acids of similar properties are often referred to in the art as families of amino acids with similar side chains, which are well defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, lactic acid, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). As another example, the addition of one or more amino acids at the amino-and/or carboxy-terminus typically does not alter the function of the polypeptide or protein. Conservative amino acid substitutions for many commonly known non-genetically encoded amino acids are known in the art. Conservative substitutions of other non-coding amino acids may be determined based on a comparison of their physical properties with those of the genetically encoded amino acid.

Variant forms of the polypeptide include homologous sequences, conservative variants, allelic variants, natural mutants, and induced mutants.

The invention also relates to polynucleotide sequences encoding GhTMK of the invention 1 or variants thereof. The polynucleotide may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. NCBI numbers of GhTMK genes are Gh_A13G0257 and/or Gh_D13G0274, and CDS sequences of the NCBI genes are shown as SEQ ID NO. 3 and/or SEQ ID NO. 4.

Fragments, analogs, and derivatives of the polypeptides. Variants of the polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. Such nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the encoded polypeptide. A "polynucleotide encoding a polypeptide" may be a polynucleotide that includes a polynucleotide encoding the polypeptide, or may also include additional coding and/or non-coding sequences.

The GhTMK nucleotide full-length sequence of the present invention or a fragment thereof can be generally obtained by a PCR amplification method, a recombinant method or an artificial synthesis method. For the PCR amplification method, primers can be designed according to the nucleotide sequences disclosed in the present invention, particularly the open reading frame sequences, and amplified to obtain the relevant sequences using a commercially available DNA library or a cDNA library prepared according to a conventional method known to those skilled in the art as a template. When the sequence is longer, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order. Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. It is usually cloned into a vector, transferred into a cell, and then isolated from the proliferated host cell by a conventional method to obtain the relevant sequence.

Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. At present, it is already possible to obtain the DNA sequences encoding the proteins of the invention (or fragments or derivatives thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. In addition, mutations can be introduced into the protein sequences of the invention by chemical synthesis.

The invention also provides a recombinant vector comprising the gene of the invention. As a preferred mode, the promoter downstream of the recombinant vector comprises a multiple cloning site or at least one cleavage site. When it is desired to express the gene of interest of the present invention, the gene of interest is ligated into a suitable multiple cloning site or cleavage site, thereby operably linking the gene of interest to a promoter. As another preferred mode, the recombinant vector comprises (from 5 'to 3') a promoter, a gene of interest, and a terminator. The recombinant vector may further comprise, if desired, elements selected from the group consisting of 3' polynucleotide signals, untranslated nucleic acid sequences, transport and targeting nucleic acid sequences, resistance selection markers (dihydrofolate reductase, neomycin resistance, hygromycin resistance, and green fluorescent protein, etc.), enhancers, or operators.

Methods for preparing recombinant vectors are well known to those of ordinary skill in the art. The expression vector may be a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector. In general, any plasmid or vector may be used as long as it is capable of replication and stability in a host.

One of ordinary skill in the art can construct expression vectors containing the genes of the present invention using well known methods. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. When the gene of the present invention is used to construct recombinant expression vectors, any one of enhanced, constitutive, tissue-specific or inducible promoters may be added before the transcription initiation nucleotide.

Vectors comprising the genes of the invention may be used to transform an appropriate host cell to allow the host to express the protein. The host cell may be a prokaryotic cell such as E.coli, streptomyces, agrobacterium, or a lower eukaryotic cell such as a yeast cell, or a higher eukaryotic cell such as a plant cell, preferably a Malvaceae plant cell, more preferably a upland cotton cell. It will be clear to one of ordinary skill in the art how to select appropriate vectors and host cells. Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote (e.g., E.coli), the treatment may be performed by CaCl ₂ or electroporation. When the host is eukaryotic, DNA transfection methods such as calcium phosphate co-precipitation, conventional mechanical methods (e.g., microinjection, electroporation, liposome packaging, etc.) may be used. The transformed plant may also be transformed by Agrobacterium or gene gun, such as leaf disc method, embryo transformation method, flower bud soaking method, etc. Plants can be regenerated from the transformed plant cells, tissues or organs by conventional methods to obtain transgenic plants. When expressed in higher eukaryotic cells, the polynucleotide will have enhanced transcription if inserted into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs, that act on a promoter to increase the transcription of a gene.

The invention also provides a method of constructing a transgenic plant comprising regenerating a host cell containing a polypeptide or polynucleotide described herein into a plant, said host cell being a plant cell. Methods and reagents for regenerating plant cells are well known in the art.

The invention also provides a method of constructing a transgenic plant comprising transforming a plant with a polynucleotide or nucleic acid construct as described herein, and obtaining a transgenic positive plant expressing a polypeptide as described herein, comprising said polynucleotide or comprising said nucleic acid construct in the progeny of the plant by crossing, screening. Methods for transforming plants with nucleic acids, crossing plants, and screening for transgenic positive plants are well known in the art.

The invention also provides a method for regulating plant fiber cell elongation or regulating plant fiber length, comprising (1) up-regulating GhTMK protein expression or activity in a plant, thereby promoting plant fiber cell elongation or up-regulating plant fiber length, or (2) down-regulating GhTMK protein expression or activity in a plant, thereby inhibiting plant fiber cell elongation or down-regulating plant fiber length,

In one or more embodiments, (1) comprises transferring the coding sequence of GhTMK1 protein into a plant to obtain a transformed plant, (2) comprises (a) specifically interfering with GhTMK1 gene transcription and/or expression, (b) down-regulating GhTMK1 protein activity, or (c) expressing GhTMK protein with reduced activity in a plant, in one or more embodiments, (a) wherein the interfering is interfering with GhTMK1 gene transcription or translation of its transcript, (b) wherein the down-regulating GhTMK1 protein activity comprises in a plant, (i) expressing a specific antibody or ligand (e.g., an inhibitory antibody) capable of down-regulating GhTMK protein of GhTMK protein activity, or (ii) introducing a nucleic acid sequence encoding (i) and/or a nucleic acid construct capable of expressing (i)

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein. The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

Examples

EXAMPLE 1 GhTMK1 full-length Gene cloning

We analyzed and screened the upland cotton annotated TMK gene. Upland cotton is found to have 3 pairs of TMK genes which are distributed on chromosome 11, chromosome 12 and chromosome 13 respectively. These 3 pairs of TMKs are most similar to Arabidopsis TMK1. TMK and Arabidopsis TMK1 similarity on chromosomes 11, 12, and 13 were 76%, 61%, and 62%, respectively (FIG. 1A). We named GhTMK1 as a pair of TMKs (Gh_A13G0257, gh_D13G0274) on chromosome 13.

A. cotton RNA extraction (RNAprep Pure polysaccharide polyphenol plant total RNA extraction kit, tiangen, beijing)

(1) Taking appropriate amount of ovule with fiber 6 days after flowering, grinding in liquid nitrogen containing filter screen, separating ovule and fiber, grinding fiber into powder, and placing into 2mL EP tube. Add 0.7mL of SL cleavage

The liquid was thoroughly vortexed.

(2) Centrifuge at 13,500g for 2min.

(3) The supernatant was transferred to a filter column CS, centrifuged for 2min at 13,500g, and carefully aspirated the supernatant from the collection tube into a new RNase-Free centrifuge tube.

(4) Slowly adding 0.4 times of absolute ethyl alcohol with the supernatant volume, uniformly mixing, transferring the obtained solution and the precipitate into an adsorption column CR3, centrifuging 13,500g for 15sec, pouring out waste liquid in a collecting pipe, and placing the adsorption column CR3 back into the collecting pipe.

(5) 350. Mu.L of deproteinized liquid RW1,13,500g was added to the adsorption column CR3, centrifuged for 15sec, and the waste liquid in the collection tube was discarded, and the adsorption column CR3 was returned to the collection tube.

(6) Preparing DNase I working solution, namely placing 10 mu L of DNase I storage solution into a new RNase-Free centrifuge tube, adding 70 mu L of RDD buffer solution, and gently mixing.

(7) 80. Mu.L of DNase I working solution was added to the center of the column CR3 and left at room temperature for 15min.

(8) 350. Mu.L of deproteinized liquid RW1,13,500g was added to the adsorption column CR3, centrifuged for 15sec, and the waste liquid in the collection tube was discarded, and the adsorption column CR3 was returned to the collection tube.

(9) To the adsorption column CR3, 500. Mu.L of the rinse liquid RW was added, and 13,500g was centrifuged for 15sec, and the waste liquid in the collection tube was poured off, and the adsorption column CR3 was returned to the collection tube.

(10) Step 9 is repeated.

(11) 13,500G of the solution is centrifuged for 2min, the adsorption column CR3 is placed in a new RNase-Free centrifuge tube, 50 mu L of RNase-Free ddH ₂ O is suspended and dripped into the middle part of the adsorption film, 2min is placed at room temperature, and 13,500g of the solution is centrifuged for 1min, so that an RNA solution is obtained.

B. Reverse transcription (TRANSSCRIPT ONE-Step RT-PCR Supermix kit, transgene, shanghai)

The concentration of the extracted RNA was measured using Nanodrop. To the reaction system, 1. Mu.L of reverse transcriptase, 1. Mu.L of oligo Dt, 1. Mu.L of gDNA reverse, 2. Mu.L of 10 Xreaction buffer, 2. Mu.g of RNA were added, and the system was made up to 20. Mu.L with ddH ₂ O. 42℃for 30min and 85℃for 5min, and 4℃to give a reverse transcription product.

PCR cloning GhTMK Gene CDS sequence

Based on the GhTMK a cDNA sequence, specific primers were synthesized:

GhTMK1-F:5'-ATGGCGAATACCCTTCCCTTTG-3'

GhTMK1-R:5'-TCATCGAGCATCAGCTGATGCG-3'

The reverse transcription product of the total RNA of the 6DPA fiber is used as a template and is subjected to PCR reaction by using high-fidelity enzyme under the reaction conditions of 98 ℃ presaturation for 5min, 98 ℃ denaturation for 30s,56 ℃ renaturation for 30s and 72 ℃ extension for 2min, 38 cycles in total and 72 ℃ extension for 10min. The PCR products were purified by electrophoresis, recovered, subcloned into commercial T-carriers and sequenced to confirm correct sequences.

Example 2 characterization of GhTMK1 expression in various tissues of upland cotton

Taking roots, stems and leaves of seedlings growing for 4 weeks from wild R15, separating ovules, petals, stamens and pistils from flowers on the day of flowering, collecting fibers 3, 6, 9,12, 15, 18 and 21 days after flowering, quick-freezing with liquid nitrogen, grinding, extracting RNA, carrying out reverse transcription, and diluting the obtained reverse transcription product by 2 times. And designing GhTMK1 detection primers, and carrying out qRT-PCR detection GhTMK expression level by taking the diluted reverse transcription product as a template. The qRT-PCR result shows that GhTMK1 is specifically expressed in cotton fiber with high expression level and lower expression level in other tissues of cotton. During the fiber development, ghTMK1 was gradually increased in expression level during elongation, and the highest expression level was obtained 9 days after flowering (fig. 2), suggesting that GhTMK1 plays a role in regulating the elongation of the fiber cells.

Specific primer for qRT-PCR detection GhTMK of expression quantity:

q-GhTMK1-F:5'-CCATGGCAGGTTCCTGAGAG-3'

q-GhTMK1-R:5'-GGTTGTTCATGGCCACATGC-3'

Example 3,35S:: ghTMK1, 35S:: dsGhTMK1 and RDL1:: ghTMK1-YFP transgenic cotton construction

Construction of a 35S-GhTMK plant expression vector

The high-fidelity enzyme amplifies GhTMK-A CDS full-length sequence and introduces a homology arm, the GhTMK-1-A CDS full-length sequence is cloned to a 35S:: NOS/2301 vector through homologous recombination to form a 35S::: ghTMK1 plant expression vector, and the sequence is correct through sequencing verification.

Construction of a plant expression vector dsGhTMK A

Amplifying the selected GhTMK RNA interference fragment by high-fidelity enzyme, introducing a homology arm, cloning forward and reverse sequences to two ends of an RTM sequence on a pCambia2301-RTM-KY vector containing an RTM intron through homologous recombination to form a 35S: dsGhTMK1 plant expression vector, and verifying that the sequences are correct through sequencing.

Construction of RDL1: ghTMK1-YFP plant expression vector

Sequentially synthesizing RDL1 promoter (fiber specific expression promoter), ghTMK _A CDS and YFP CDS sequences, removing a stop codon at the tail end of GhTMK1_A to ensure the fusion expression of GhTMK and YFP and introducing a homology arm, cloning the synthesized sequence onto a 35S:: NOS/2301 vector to replace the 35S promoter by homologous recombination, forming an RDL1:: ghTMK1-YFP plant expression vector, and sequencing to verify that the sequence is correct.

GhTMK1 RNA interference fragment sequence:

CTCAAAACCAGCCTAAAGCTCCCTTCTTCGCTCGATTGGTCTGACCCTGACCCTTGCCAATGGACCAACGTTCGCTGTGAAAACCAGAGGATCACCCGGATTCAAATTCCCAGCAAAAACGTTGGTGGAACCCTTCCCCCTGATCTCAAAGACCTGTCTCAGCTTAAAGTCTTTGAAGTCATGAACAACCAAATCAGTGGACCCATCCCCAGCCTTGCTGGGTTAAGCCTGTTGGAAGAAGCCAACTTCCATGACAACAATTTCTCATCTTTCCCTTCTGATTTCTTCACTGGCTTAACCTCGTTAACCGCTATTTACCTCGATAACAACCCTTTCGAGCCATGGCAGGTTCCTGAGAGCATAAAGGAAGCAACTTCTTTGAAAGCTTTCTCTGCTAACAAG

d. Agrobacterium transformation

Thawing LBA4404 agrobacterium competent ice, adding about 1 mug plasmid, standing on ice after uniform mixing for 30min, quick-freezing with liquid nitrogen for 1min, carrying out heat shock for 3-5 min in a 37 ℃ water bath, placing on ice for 3min, adding a nonreactive liquid LB culture medium for resuscitation for 2-4 h, coating on a selection culture medium (Rif 20 mug/mL, gent50 mug/mL and Kan 50 mug/mL), placing in a 28 ℃ incubator, and carrying out inversion culture for 2-3 days.

E. Cotton transformation

Agrobacterium containing 35S GhTMK and 35S dsGhTMK vector plasmids were cultured on YEB bacterial medium supplemented with 50mg/L kanamycin, 100mg/L rifampicin, 300mg/L streptomycin for 2-3 days, and single colonies were inoculated into YEB liquid medium containing the same antibiotics and cultured in suspension on a shaker at 28℃at 200rpm/min overnight. The bacterial liquid is centrifuged for 10min at 4000rpm/min, the sediment is resuspended by using a 1/2MS liquid culture medium containing 30g/L glucose and 100 mu mol/L acetosyringone, and the OD ₆₀₀ value is regulated to be about 0.4-0.6 to be used as a conversion liquid for standby.

And (3) after the upland cotton R15 seeds are subjected to conventional sterilization, placing the upland cotton R15 seeds in a 1/2MS0 (1/2 MS salt+5 g/L glucose+7 g/L agar powder, pH 6.0) culture medium, germinating and culturing in the dark, and cutting the hypocotyl of the sterile seedling into about 1.0cm sections after 5-7 days to serve as a transformation explant for standby.

The explant is soaked in agrobacterium liquid for 15-20 min, transferred to a co-culture medium MSB1 (MS salt+B5 organic+30 g/L glucose+0.1 mg/L KT+0.1 mg/L2, 4-D+2.2g/L Gelrite, pH 6.0), and after dark culture for 2 days at 22 ℃, the explant is transferred to a culture medium MSB2 (MSB1+500 mg/L cephalosporin+80 mg/L kanamycin) for callus induction. Explants are subjected to induction of resistant callus, multiplication of callus and induction of embryogenic callus (culture medium MSB3: MS salt+B5 organic+30g/L glucose+2.5g/L Gelrite, pH 6.0), somatic embryogenesis (culture medium MSB4: MS salt+B5 organic+30g/L glucose+1.0 g/L asparaginyl amine+2.0 g/L glutamine+3.0 g/L Gelrite, pH 6.0; KNO ₃ in MS salt is doubled, NH ₄NO₃ is removed), and resistant test tube plantlets are regenerated. When the test-tube plantlet grows to 3-4 true leaves, transplanting the test-tube plantlet into a flowerpot, and putting the flowerpot into a climatic chamber for growth.

Example 4 molecular biological characterization of transgenic plants

PCR identification

The DNA extraction adopts a CTAB method (1) taking 1-2g of transgenic cotton plant leaves, adding steel balls, quick-freezing with liquid nitrogen, grinding, adding 0.8mL of CTAB extract, vortex mixing, placing in a 65 ℃ oven for 30min (every 10min in the middle, mixing upside down), adding 800 mu L of chloroform, gently mixing upside down, centrifuging at 12000rpm for 10min, (3) taking supernatant, adding equal volume of chloroform, gently mixing upside down, centrifuging at 12000rpm for 10min, (4) repeating the step (3), (5) taking supernatant, adding half volume of isopropanol, gently mixing upside down, standing on ice for 1h or overnight at-20 ℃, centrifuging at 12000rpm for 10min, (6) pouring supernatant, adding 75% ethanol of equal volume of isopropanol, gently mixing upside down, centrifuging at 12000rpm for 10min, (7) repeating the step (6), pouring supernatant, airing at 37 ℃ and residual ethanol. Adding 50 mu L ddH ₂ O (containing RNase) to dissolve DNA, reacting at 37 ℃ for 30min to digest RNA, and standing at 65 ℃ for 15min to inactivate RNase to obtain DNA.

Designing specific PCR identification primers, carrying out PCR reaction by taking the extracted DNA as a template, taking ddH ₂ O as a negative control as a template, and taking a corresponding vector plasmid as a template as a positive control. The PCR product was run on a nucleic acid gel to confirm whether there was a positive band of the correct size.

CTAB extract, 100mM Tris-HCl (pH 8.0), 20mM EDTA (pH 8.0), 1.4M sodium chloride, 2% CTAB (w/v), 2% beta-mercaptoethanol (v/v).

GUS staining

Adding a small amount of 35S (GhTMK S) and 35S (dsGhTMK) transgenic cotton plant leaves into a 96-well plate, adding GUS staining buffer solution until the leaves are completely immersed, pumping air in a vacuum pump for 30min, incubating at 37 ℃ for 12h, and observing whether the leaves and the buffer solution turn blue or not, and if so, judging that the corresponding plants are positive plants.

GUS staining buffer 50mM sodium phosphate (pH 7.0), 10mM EDTA (pH 8.0), 20% methanol, 1% Triton X-100,2mM potassium ferrocyanide, 2mM potassium ferricyanide, 0.2mg/mL X-Gluc.

Example 5 transgenic Cotton Gene expression and fiber trait analysis

A. Transgenic cotton gene expression analysis

Extracting RNA from 35S GhTMK S dsGhTMK S9 DPA fiber of transgenic cotton with positive PCR identification and positive GUS staining, reverse transcription, and diluting the obtained reverse transcription product by 2 times. And designing GhTMK1 detection primers, and carrying out qRT-PCR detection GhTMK expression level by taking the diluted reverse transcription product as a template. The results showed that the expression level of GhTMK1 in 35S: ghTMK1 transgenic cotton fibers was significantly increased (FIG. 4A), while the expression level of GhTMK1 in 35S: dsGhTMK1 transgenic cotton fibers was significantly decreased (FIG. 3A), indicating that the vectors transformed into cotton all played a corresponding role.

B. Transgenic cotton fiber trait analysis

35S GhTMK1, 35S dsGhTMK1 transgenic cotton and wild R15 were grown in a climatic chamber. After cotton maturing and boll opening, a single plant collects mature cotton bolls, a certain number of seeds are randomly taken, fibers of the cotton bolls are combed flat, fiber lengths of the cotton bolls are measured, and statistical analysis is carried out. The results showed that 35S dsGhTMK1 transgenic cotton mature fiber length was significantly reduced (FIGS. 3B, C), while 35S GhTMK1 transgenic cotton mature fiber length was significantly increased (FIGS. 4B, C), indicating that high expression of GhTMK1 in cotton fiber cells has a promoting effect on elongation growth, and enhanced expression of GhTMK1 can promote elongation growth of fiber cells.

Example 6 observation of subcellular localization of GhTMK1 protein

GhTMK1-YFP transgenic cotton 1DPA ovules are taken and cut, and YFP signals in the fibroblasts are observed under a fluorescence confocal microscope. The results showed that YFP signals were distributed on the fibrous cell membrane and accumulated at the fibrous cell tip, indicating that GhTMK a1 polarity was localized at the fibrous cell membrane tip (fig. 5). GhTMK1 accumulation at the tip of the fiber cells may result in a greater growth rate at the tip of the fiber cells than elsewhere, thereby promoting polar elongation of the fiber cells.

Partial sequences herein

SEQ ID NO:1>Gh_A13G0257

MANTLPFGGGCFLFGFLSVLCFFALHVSSQSSPDSSVMGKLKTSLKLPSSLDWSDPDPCQWTNVRCENQRITRIQ

IPSKNVGGTLPPDLKDLSQLKVFEVMNNQISGPIPSLAGLSLLEEANFHDNNFSSFPSDFFTGLTSLTAIYLDNNPF

GPWQVPESIKEATSLKAFSANKANLNGRFPGLFDLATFPGLTDLHVAMNNLEGELPASLAGSMIQSFWANGQK

LNGTIEVIQNMSSLREVWLNMNQFRGPLPDFSMLTQLSNLSLRDNQLTGVVPSSLMNLKSLYIVNLTNNKLQGP

TPKFANGVILDMRAGSNRFCLDDPGVACDERVTILLSIMEAFGYPENFADNWKGNDPCNDWLGISCVQGNIVSI

LFAKKGLTGTISSNFAKLDSLTTLDLSGNNLTGTIPTELTTLPKLVRLDVSNNRLYGKVPPFRQNVAVITAGNPDI

GKEMASPPAGKSPGGSPAGGGGSSGSSSGNGEKKSNTGTVVGSAIGAVGGLSLLVLGILLYARKGKRTSKVQSP

TTVVIHPHHSDDQDGVKITVAGSSVTGGSETFSHTSSGPSDVHLVEAGNMVISIQVLRDVTNNFSEENVLGRGGF

GTVYKGELHDGTKIAVKRMESSVVSEKGLAEFKSEIAVLTKVRHRHLVALLGYCLDGNERLLVYEYMPQGTLS

RHLFNWKHEGLKPLEWTRRLTIALDVARGVEYLHGLAQQSFIHRDLKPSNILLGDDMRAKVADFGLVRLAPVD

GKQSIETRLAGTFGYLAPEYAVTGRVTTKVDVFSFGVILMELISGRRALDETQPEESMHLVSWFRRMHMNKETF

RKAIDETIQLDEETLASVSTVTELAGHCCAREPYQRPDMSHAVNVLSSLAELWKPAEPDSDDIYGIDLELTLPQA

LKKWQAFEGNSNVDDSSSFLGSTDTTQTSIPCRPPGFADSFASADAR

SEQ ID NO:2>Gh_D13G0274

MANTLPFGGGCFLFGFLSVLCFFALHVSSQSSPDSSVMGKLKTSLKLPSSLDWSDPDPCQWTNVRCENQRITRIQ

IPSKNVGGTLPPDLKDLSQLKVFEVMNNQISGPIPSLAGLSLLEEANFHDNNFSSFPSDFFTGLTSLTAIYLDNNPF

EPWQVPESIKEATSLKAFSANKANLNGRFPGLFDLATFPGLTDLHVAMNNLEGELPASLAGSMIQSFWANGQRL

NGTIEVIQNMSSLREVWLNMNQFTGPLPDFSMLTQLSNLSLRDNQLTGVVPSSLINLKSLYIVNLTNNKLQGPTP

KFADGVILDMRAGSNRFCLDDPGVACDERVTILLSIMEAFGYPENFADNWKGNDPCNDWLRISCVQGNIVSILF

AKKGLTGTISSNFAKLDSLTTLDLSGNNLTGTIPTELTTLPKLVRLDVSNNRLHGKVPPFRQNVAVITAGNPDIGK

EMASPPSGKSPGGSPGGGGGGGSSSGNGEKKLNTGTVVGSAIGAVGGLSLLVLGICLYARKGKRTSKVRSPATV

VIHPHHSGDQDGVKITVAGSSVTGGSETFSHSSSGPTDVHLVEAGNMVISIQVLRDVTNNFSEENVLGRGGFGTV

YKGELHDGTKIAVKRMESSVVSEKGLAEFKSEIAVLTKVRHRHLVALLGYCLDGNERLLVYEYMPQGTLSRHL

FNWKHEGLKPLEWTRRLTIALDVARGVEYLHGLAQQSFIHRDLKPSNILLGDDMRAKVADFGLVRLAPVDGKQ

SIETRLAGTFGYLAPEYAVTGRVTTKVDVFSFGVILMELISGRRALDETQPEESMHLVSWFRRMHMNKDTFRKA

IDETIQLDEETLASVSTVTELAGHCCAREPYQRPDMSHAVNVLSSLAELWKPAEPDSDDIYGIDLELTLPQALKK

WQAFEGNSSVDDSSSFLGSTDTTQTSIPCRPPGFADSFASADAR

SEQ ID NO:3>Gh_A13G0257

ATGGCGAATACCCTTCCCTTTGGGGGTGGTTGTTTTCTTTTTGGGTTTCTTTCAGTTCTTTGCTTCTTTGCTTT

ACATGTCTCTTCTCAATCCAGCCCTGATTCTTCAGTCATGGGAAAACTCAAAACCAGCCTAAAGCTCCCTTC

TTCGCTCGATTGGTCTGACCCTGACCCTTGCCAATGGACCAACGTTCGCTGTGAAAACCAGAGGATCACCC

GGATTCAAATTCCCAGCAAAAACGTTGGAGGAACCCTTCCCCCTGATCTCAAAGACCTGTCTCAGCTTAAA

GTCTTTGAAGTCATGAACAACCAAATCAGTGGACCCATCCCGAGCCTTGCTGGGTTAAGCCTGTTGGAAGA

AGCCAACTTCCATGACAACAATTTCTCATCTTTCCCTTCTGATTTCTTCACTGGCTTAACCTCGTTAACCGCT

ATTTACCTCGATAACAACCCTTTCGGGCCATGGCAGGTTCCTGAGAGCATAAAGGAAGCGACTTCTTTGAA

AGCTTTCTCTGCTAACAAGGCTAATCTTAATGGGAGATTCCCAGGTTTGTTTGACCTAGCTACTTTTCCAGG

CTTGACAGATTTGCATGTGGCCATGAACAACCTTGAAGGTGAATTGCCTGCATCCTTAGCGGGTTCTATGAT

TCAGTCTTTCTGGGCTAATGGGCAGAAGTTGAATGGGACAATTGAAGTGATACAGAACATGTCTTCTTTAA

GAGAGGTATGGTTGAATATGAACCAGTTTAGAGGTCCCTTGCCTGATTTTTCAATGTTGACTCAGTTGAGCA

ATTTGAGTTTGAGGGATAATCAGCTCACTGGGGTTGTTCCTTCGTCTTTGATGAACTTAAAGTCACTCTATA

TCGTGAATTTGACTAATAATAAACTTCAAGGGCCAACCCCTAAATTTGCTAATGGTGTAATTCTAGATATGA

GGGCAGGTAGTAATAGATTTTGCTTGGATGATCCTGGTGTTGCTTGTGATGAACGTGTTACTATTTTGTTGT

CTATAATGGAAGCTTTTGGTTATCCAGAGAATTTTGCTGATAATTGGAAGGGGAATGATCCTTGTAATGACT

GGTTAGGTATTTCATGTGTTCAAGGAAATATTGTGTCTATCCTTTTTGCAAAAAAAGGGCTTACTGGAACTA

TTTCTAGTAATTTTGCGAAGCTTGATTCCTTGACAACTTTGGATCTTTCGGGTAATAATCTTACTGGCACAAT

ACCAACAGAGCTCACTACATTGCCGAAGCTTGTTCGATTAGATGTTTCGAACAACAGGCTCTATGGAAAAG

TACCACCTTTCAGGCAAAATGTGGCGGTGATAACTGCTGGTAACCCTGATATAGGAAAAGAAATGGCTTCT

CCACCAGCTGGCAAGTCCCCTGGTGGATCTCCTGCTGGCGGCGGCGGCAGCAGCGGCAGTTCCTCTGGAAA

TGGTGAAAAGAAATCGAATACGGGGACTGTTGTGGGTTCTGCAATTGGTGCGGTTGGTGGCTTGAGTCTTC

TTGTTTTGGGTATCCTTTTGTATGCTAGAAAAGGAAAGCGTACCAGTAAAGTGCAGAGTCCAACTACTGTA

GTCATACACCCTCATCATTCCGATGACCAGGATGGAGTTAAAATCACTGTCGCTGGATCAAGTGTCACTGG

TGGAAGTGAAACTTTCAGCCACACGAGCAGTGGACCAAGTGATGTTCACTTGGTTGAGGCTGGCAACATGG

TGATCTCGATTCAAGTTTTGAGGGATGTGACAAATAATTTCAGTGAGGAAAATGTGTTAGGAAGGGGTGGT

TTTGGGACAGTTTACAAAGGGGAATTACATGATGGGACAAAGATTGCTGTGAAAAGGATGGAGTCCAGTG

TTGTGAGCGAGAAGGGTTTGGCCGAGTTTAAGTCCGAGATTGCAGTTCTTACTAAGGTTCGTCACCGCCATT

TAGTTGCACTTCTTGGATATTGCTTGGATGGAAATGAGAGGCTTCTTGTATATGAATATATGCCTCAAGGGA

CCCTTAGTCGGCATCTATTCAACTGGAAACATGAAGGTCTGAAACCACTTGAATGGACTAGACGACTTACA

ATTGCCCTAGATGTCGCTCGAGGTGTTGAGTATCTACATGGTCTGGCACAACAGAGTTTCATTCATCGAGAT

CTTAAGCCGTCAAATATTCTTCTCGGAGACGATATGCGTGCCAAGGTTGCAGATTTTGGCTTGGTCCGTCTA

GCTCCTGTGGATGGCAAACAGTCTATTGAAACAAGACTAGCAGGAACCTTTGGGTATTTGGCACCGGAGTA

TGCAGTTACTGGACGAGTAACCACGAAGGTAGATGTGTTTAGCTTTGGTGTGATCCTCATGGAATTGATCTC

AGGCCGAAGGGCACTTGATGAAACTCAGCCTGAGGAAAGCATGCACCTTGTGTCATGGTTCCGTCGGATGC

ACATGAACAAAGAAACCTTCCGGAAGGCCATTGATGAAACAATCCAGCTTGATGAGGAAACGCTAGCCAG

TGTTAGCACAGTGACTGAGCTTGCTGGCCACTGTTGTGCCAGGGAGCCCTACCAGAGGCCAGATATGAGCC

ATGCGGTCAATGTGCTGTCATCACTAGCAGAGCTCTGGAAACCAGCAGAACCCGATTCCGACGACATCTAT

GGCATTGACCTTGAATTAACTTTACCCCAAGCATTGAAAAAGTGGCAAGCCTTCGAGGGGAACAGCAATGT

GGATGACTCCTCATCATTTCTGGGAAGTACAGACACTACACAGACCAGCATACCATGCCGGCCGCCAGGTT

TTGCTGATTCATTCGCATCAGCTGATGCTCGATGA

SEQ ID NO:4>Gh_D13G0274

ATGGCGAATACCCTTCCCTTTGGGGGTGGTTGTTTTCTTTTTGGGTTTCTTTCAGTTCTTTGCTTCTTTGCTTT

ACATGTCTCTTCTCAATCCAGCCCTGATTCTTCAGTCATGGGAAAACTCAAAACCAGCCTAAAGCTCCCTTC

TTCGCTCGATTGGTCTGACCCTGACCCTTGCCAATGGACCAACGTTCGCTGTGAAAACCAGAGGATCACCC

GGATTCAAATTCCCAGCAAAAACGTTGGTGGAACCCTTCCCCCTGATCTCAAAGACCTGTCTCAGCTTAAA

GTCTTTGAAGTCATGAACAACCAAATCAGTGGACCCATCCCCAGCCTTGCTGGGTTAAGCCTGTTGGAAGA

AGCCAACTTCCATGACAACAATTTCTCATCTTTCCCTTCTGATTTCTTCACTGGCTTAACCTCGTTAACCGCT

ATTTACCTCGATAACAACCCTTTCGAGCCATGGCAGGTTCCTGAGAGCATAAAGGAAGCAACTTCTTTGAA

AGCTTTCTCTGCTAACAAGGCTAATCTTAATGGGAGATTCCCAGGTTTGTTTGACCTTGCTACTTTTCCAGG

CTTGACAGATTTGCATGTGGCCATGAACAACCTTGAAGGTGAATTGCCTGCATCCTTAGCGGGTTCCATGAT

TCAGTCTTTCTGGGCCAATGGGCAGAGGTTGAATGGGACAATTGAAGTGATACAGAACATGTCTTCTTTAA

GAGAGGTATGGTTGAATATGAACCAGTTTACAGGTCCCTTGCCTGATTTTTCAATGTTGACTCAGTTGAGCA

ATTTGAGTTTGAGGGATAATCAGCTCACTGGTGTTGTTCCTTCGTCTTTGATTAACTTAAAGTCACTCTATAT

CGTGAATTTGACTAATAATAAACTTCAAGGACCAACCCCTAAATTTGCTGATGGTGTAATTCTAGATATGA

GGGCTGGTAGTAATAGATTTTGCTTGGATGATCCTGGTGTTGCTTGTGATGAACGTGTTACTATTTTATTGT

CTATAATGGAAGCTTTTGGTTATCCAGAGAATTTTGCTGATAATTGGAAGGGGAATGATCCTTGTAATGACT

GGTTACGTATTTCATGTGTTCAAGGAAATATTGTGTCTATCCTTTTTGCAAAAAAAGGGCTTACTGGTACTA

TTTCTAGTAATTTTGCGAAGCTTGATTCCTTGACAACTTTGGATCTTTCCGGTAATAATCTTACCGGCACGAT

ACCGACAGAGCTCACTACATTGCCAAAGCTTGTTCGATTAGATGTTTCGAACAACAGGCTACATGGAAAAG

TACCACCTTTCAGGCAAAATGTGGCGGTGATAACTGCTGGTAACCCTGATATAGGAAAAGAAATGGCTTCT

CCACCATCTGGCAAGTCCCCTGGTGGATCTCCTGGCGGCGGCGGCGGCGGCGGTAGTTCCTCTGGAAATGG

TGAAAAGAAATTGAATACGGGGACAGTTGTGGGTTCTGCAATTGGTGCAGTTGGTGGCTTGAGTCTTCTTG

TTTTGGGTATCTGTTTGTATGCTAGAAAAGGAAAGCGTACCAGTAAAGTGCGGAGTCCAGCTACTGTAGTC

ATACATCCTCATCATTCCGGTGACCAAGATGGAGTTAAAATCACCGTTGCTGGATCGAGCGTCACTGGTGG

AAGTGAAACTTTCAGCCACTCAAGCAGTGGACCAACTGATGTTCACTTGGTTGAGGCTGGCAACATGGTGA

TCTCGATTCAAGTTTTGAGGGATGTGACAAATAATTTCAGTGAGGAAAATGTGCTAGGAAGGGGTGGTTTT

GGGACAGTTTACAAAGGGGAATTACATGATGGGACAAAGATTGCGGTGAAAAGGATGGAGTCCAGTGTTG

TGAGCGAGAAGGGTTTGGCCGAGTTTAAGTCCGAGATTGCAGTTCTTACTAAGGTTCGTCACCGCCATTTA

GTCGCACTTCTTGGATATTGCTTGGATGGAAATGAGAGGCTTCTTGTATATGAATATATGCCTCAAGGGACC

CTTAGTCGGCACCTATTCAACTGGAAACATGAAGGACTGAAACCACTTGAATGGACTAGACGACTTACAAT

TGCCCTAGATGTCGCTCGAGGTGTTGAGTATCTACATGGTCTGGCACAACAGAGTTTCATTCATCGAGATCT

TAAGCCATCAAATATTCTTCTCGGAGATGATATGCGTGCCAAGGTTGCAGATTTTGGCTTGGTCCGTCTAGC

TCCTGTGGATGGCAAACAGTCAATTGAAACAAGGCTAGCAGGAACCTTTGGGTATTTGGCACCGGAGTATG

CAGTTACTGGACGAGTAACCACGAAGGTAGATGTATTTAGCTTTGGTGTGATCCTCATGGAATTGATCTCA

GGCCGAAGGGCACTTGATGAAACTCAGCCCGAGGAAAGCATGCACCTTGTGTCATGGTTCCGTCGGATGCA

CATGAACAAAGACACATTCCGGAAGGCCATTGACGAAACAATCCAGCTTGATGAGGAAACGCTAGCCAGT

GTTAGCACAGTGACTGAGCTTGCTGGCCACTGTTGTGCTAGGGAGCCCTACCAGAGGCCAGATATGAGCCA

TGCGGTCAATGTGCTGTCATCACTAGCAGAGCTCTGGAAACCAGCAGAACCCGATTCGGATGACATCTATG

GCATTGACCTTGAATTAACTTTACCCCAGGCATTGAAAAAGTGGCAAGCCTTCGAGGGGAACAGCAGTGTG

GATGACTCCTCATCATTTCTGGGCAGTACAGACACTACACAGACCAGCATACCATGCAGGCCGCCAGGTTT

TGCTGATTCATTCGCATCAGCTGATGCCCGATGA

SEQ ID NO:5

CTCAAAACCAGCCTAAAGCTCCCTTCTTCGCTCGATTGGTCTGACCCTGACCCTTGCCAATGGACCAACGTT

CGCTGTGAAAACCAGAGGATCACCCGGATTCAAATTCCCAGCAAAAACGTTGGTGGAACCCTTCCCCCTGA

TCTCAAAGACCTGTCTCAGCTTAAAGTCTTTGAAGTCATGAACAACCAAATCAGTGGACCCATCCCCAGCC

TTGCTGGGTTAAGCCTGTTGGAAGAAGCCAACTTCCATGACAACAATTTCTCATCTTTCCCTTCTGATTTCTT

CACTGGCTTAACCTCGTTAACCGCTATTTACCTCGATAACAACCCTTTCGAGCCATGGCAGGTTCCTGAGAG

CATAAAGGAAGCAACTTCTTTGAAAGCTTTCTCTGCTAACAAG。

Claims (10)

1. A nucleic acid construct comprising

(1) Nucleic acid sequence encoding GhTMK protein, or

(2) Nucleic acid sequences which specifically interfere with the transcription and/or expression of GhTMK genes,

Preferably, the amino acid sequence of GhTMK1 protein is shown as SEQ ID NO.1 or 2 or a sequence with at least 80% identity, and NCBI number of GhTMK1 gene is Gh_A13G0257 and/or Gh_D13G0274.

2. The nucleic acid construct of claim 1, wherein,

The nucleic acid sequence for encoding GhTMK protein is shown as SEQ ID NO 3 or 4 or a sequence with at least 80% identity with the same, and/or

(2) The nucleic acid sequence is shown as SEQ ID NO. 5 or a sequence having at least 80% identity thereto, and/or

The nucleic acid construct further comprises a promoter operably linked to the nucleic acid sequence,

Preferably, the nucleic acid construct has elements from 5 'to 3' such as a promoter, a nucleic acid sequence encoding GhTMK protein, and a terminator.

3. A genetically engineered host cell comprising a host cell, which is a kind of

(1) Expression, inclusion or secretion of GhTMK proteins, or

(2) Comprising the nucleic acid construct of claim 1,

The host cell is not a plant cell,

Preferably, the GhTMK protein has the amino acid sequence shown as SEQ ID NO. 1 or 2 or a sequence having at least 80% identity thereto.

4. A method of modulating plant fiber cell elongation or modulating plant fiber length, the method comprising:

(1) Up-regulating GhTMK protein expression or activity in plants to promote plant fiber cell elongation or up-regulating plant fiber length, or

(2) Down-regulating GhTMK protein expression or activity in plant, so as to inhibit plant fibre cell elongation growth or down-regulate plant fibre length,

Preferably, the method comprises the steps of,

The up-regulating of expression in GhTMK protein in plant includes transferring GhTMK protein coding sequence into plant to obtain transformed plant,

The downregulation of GhTMK protein expression or activity in plants includes (a) specific interference with GhTMK gene transcription and/or expression, (b) downregulation of GhTMK protein activity, or (c) expression of a reduced activity GhTMK protein in plants,

More preferably, the process is carried out,

(A) Wherein said interference is interference with the transcription of GhTMK gene or the translation of its transcript,

(B) Wherein said down-regulating GhTMK protein activity comprises (i) expressing in a plant an antibody or ligand specific for a GhTMK protein capable of down-regulating GhTMK protein activity (e.g., an inhibitory antibody), or (ii) introducing a nucleic acid sequence encoding (i) and/or a nucleic acid construct capable of expressing (i).

5. The method of claim 4, wherein the plant is a malvaceae plant, preferably the plant is a cotton plant.

6. The use of a substance that modulates GhTMK protein expression or activity in plants to modulate plant fiber cell elongation or modulate plant fiber length,

Preferably, the agent is an enhancer of GhTMK protein expression or activity, the modulation being upregulation of GhTMK protein expression or activity in the plant, thereby enhancing plant fiber cell elongation or upregulation of plant fiber length, more preferably, the enhancer is GhTMK protein or coding sequence thereof,

Preferably, the agent is an inhibitor of GhTMK protein expression or activity, which is down-regulating GhTMK protein expression or activity in a plant, thereby inhibiting plant fiber cell elongation or down-regulating plant fiber length, more preferably, the inhibitor is selected from (1) an inhibitor that specifically interferes with GhTMK gene transcription and/or expression, (2) an inhibitor that down-regulates GhTMK protein activity, or (3) a GhTMK protein variant or coding sequence thereof that down-regulates activity.

7. The use according to claim 6, wherein,

(1) The inhibitor is selected from the group consisting of (i) an antisense nucleic acid, microRNA, siRNA, shRNA, dsRNA, sgRNA, or a combination thereof, and (ii) a nucleic acid construct capable of expressing or forming (i),

(2) The inhibitor is selected from the group consisting of (i) antibodies or ligands specific for GhTMK protein, and (ii) nucleic acid sequences encoding (i) and/or nucleic acid constructs capable of expressing (i),

Preferably, the inhibitor of (1) is a nucleic acid that specifically interferes with the transcription and/or expression of the GhTMK gene, the sequence of which is shown as SEQ ID NO 5 or a sequence having at least 80% identity thereto.

8. A method for screening plants for fiber length, which comprises comparing the expression or activity of plant GhTMK1 gene with that of wild type plant, if GhTMK gene expression or activity is up-regulated, plant fiber cell elongation growth is enhanced or plant fiber is longer, and if GhTMK gene expression or activity is down-regulated, plant fiber cell elongation growth is inhibited or plant fiber is shorter.

9. The GhTMK gene is used as molecular marker for identifying plant fiber length,

Preferably, the steps of comparing the expression or activity of the plant GhTMK gene to a wild-type plant are performed such that if the expression or activity of the GhTMK gene is up-regulated, the plant fiber cell elongation growth is enhanced or the plant fiber is longer, and if the expression or activity of the GhTMK gene is down-regulated, the plant fiber cell elongation growth is inhibited or the plant fiber is shorter.

10. A method of obtaining a transgenic plant comprising the steps of:

(1) Providing an Agrobacterium harboring the nucleic acid construct of claim 1 or 2,

(2) Contacting a cell or tissue or organ of a plant with the agrobacterium of step (1), thereby transferring the nucleic acid construct into the plant tissue or organ.

(3) Selecting a plant tissue, organ or seed into which has been transferred a nucleic acid sequence that is (a) a nucleic acid sequence encoding a GhTMK protein, or (b) a nucleic acid sequence that specifically interferes with transcription and/or expression of a GhTMK gene, and

(4) Regenerating the plant tissue, organ or seed of step (3).