CN108623665B

CN108623665B - Application of GhHUB2 protein in regulation of cotton fiber length and strength

Info

Publication number: CN108623665B
Application number: CN201810455552.XA
Authority: CN
Inventors: 董江丽; 王涛; 冯浩
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2018-05-14
Filing date: 2018-05-14
Publication date: 2020-08-18
Anticipated expiration: 2038-05-14
Also published as: CN108623665A

Abstract

The invention discloses application of GhHUB2 protein in regulation and control of length and strength of cotton fibers. The invention provides application of GhHUB2 protein or a coding gene thereof in any one of the following methods: regulating and controlling the length of cotton fibers; regulating and controlling the strength of cotton fibers; regulating the cell wall thickness of cotton fibers; regulating and controlling the micronaire value of the cotton fiber. The invention constructs an overexpression vector of GhHUB2 gene, transforms cotton seed 24 in a receptor material by utilizing an agrobacterium-mediated genetic transformation method to obtain stably inherited transgenic cotton, and obtains a new cotton seed with increased fiber length and enhanced strength by detecting the quality of cotton fiber. The invention has important significance for cultivating new cotton varieties with better fiber quality.

Description

Application of GhHUB2 protein in regulation of cotton fiber length and strength

Technical Field

The invention relates to the technical field of biology, and in particular relates to application of GhHUB2 protein in regulation and control of length and strength of cotton fibers.

Background

Cotton belongs to the genera Gossypium, Malvaceae, Malvales, Dicotyledoneae, angiosperma, and Dicotyledoneae. The most widespread use of cotton is in the production of fiber products. The cotton fiber can be used for manufacturing comfortable clothes, fine home decorations, durable paper money, novel plastics, electronic screens and the like. China mainly plants upland cotton, the annual cotton yield is 430 ten thousand tons, accounts for 22.3 percent of the world cotton yield, and is an important cotton production country. However, the quality of cotton fibers in China, particularly the indexes such as fiber length, strength and fineness are poor, and long stapled cotton suitable for spinning is lacked. China still needs to import about 300 ten thousand tons of high-quality raw cotton every year. Therefore, the method improves the fiber quality, increases the fiber yield, cultivates high-quality cotton varieties suitable for China, and has important economic value and strategic significance.

However, most important characters such as the quality, the yield and the resistance of cotton fibers are quantitative characters, and negative correlation exists among the characters, so that the traditional breeding method is difficult to realize the synergistic improvement of the fiber quality, the fiber yield and the resistance of cotton. In addition, the defects of long period, unstable progeny character and the like of the traditional breeding means make the conventional breeding of cotton face huge difficulties. Compared with the conventional traditional breeding, the genetic engineering technology has the advantages of short period, rapid polymerization of various oriented excellent character genes, no restriction of incompatibility of interspecific hybridization and the like, and provides an effective way for breeding high-yield and high-quality cotton varieties.

Disclosure of Invention

The invention aims to provide a new application of GhHUB2 protein and a method for cultivating transgenic cotton with improved fiber length and strength.

In a first aspect, the invention claims the application of the GhHUB2 protein or the coding gene thereof in any one of the following:

(a1) regulating cotton fiber length (such as increasing cotton fiber length);

(a2) regulating cotton fiber strength (such as enhancing cotton fiber strength);

(a3) regulating the cell wall thickness of cotton fiber (such as cell wall thickness thickening of cotton fiber);

(a4) and regulating the micronaire value of the cotton fiber (such as increasing the micronaire value of the cotton fiber).

In the present invention, all of the GhHUB2 proteins represent the cotton-derived GhHUB2 protein.

In the application, the higher the expression level and/or activity of the GhHUB2 protein or the coding gene thereof in the cotton, the longer the fiber length of the cotton, the stronger the fiber strength, the thicker the fiber cell wall thickness and/or the higher the micronaire value of the fiber; the lower the expression level and/or activity of the GhHUB2 protein or the coding gene thereof in the cotton, the shorter the fiber length of the cotton, the weaker the fiber strength, the thinner the fiber cell wall thickness and/or the lower the micronaire value of the fiber.

In a second aspect, the present invention claims a method for breeding cotton having at least one of the traits shown in (b1) - (b4), comprising the step of increasing the expression level and/or activity of GhHUB2 protein in recipient cotton;

(b1) the length of the fiber is increased;

(b2) the strength of the fiber is enhanced;

(b3) thickening the thickness of the fiber cell wall;

(b4) the micronaire value of the fibres increases.

In a third aspect, the present invention claims a method for breeding cotton having at least one of the traits shown below (c1) - (c4), comprising the step of reducing the expression level and/or activity of GhHUB2 protein in recipient cotton;

(c1) the fiber length is shortened;

(c2) the strength of the fibers is weakened;

(c3) the thickness of the fiber cell wall is reduced;

(c4) the micronaire value of the fibres decreased.

In the method of the second aspect, the increase in the expression level and/or activity of GhHUB2 protein in the cotton recipient can be achieved by introducing a gene encoding the GhHUB2 protein into the cotton recipient.

Further, this can be accomplished by any means that can accomplish this. The GhHUB2 protein coding gene can be introduced into the acceptor cotton in the form of a recombinant vector.

The recombinant vector can be constructed using existing plant expression vectors. The plant expression vector comprises a binary agrobacterium vector, a vector which can be used for plant microprojectile bombardment and the like, such as pMDC32, pCAMBIA1305.1, pCAMBIA-1300-221, pGreen0029, pCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-Ubin or other derivative plant expression vectors. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can direct the addition of poly A to the 3' end of the mRNA precursor. When the gene is used for constructing a recombinant expression vector, any one of enhanced, constitutive, tissue-specific or inducible promoters, such as a cauliflower mosaic virus (CAMV)35S promoter, a Ubiquitin gene Ubiquitin promoter (pUbi), a stress-inducible promoter rd29A and the like, can be added before the transcription initiation nucleotide, and can be used alone or in combination with other plant promoters; in addition, when the gene of the present invention is used to construct a recombinant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the recombinant expression vectors used may be processed, for example, by adding genes encoding enzymes or luminescent compounds which produce a color change, antibiotic markers having resistance or chemical resistance marker genes, etc., which are expressed in plants. Or directly screening the transformed plants in a stress environment without adding any selective marker gene.

Further, the promoter for promoting transcription of the gene in the recombinant vector may be a 35S promoter.

In the specific embodiment of the invention, the recombinant vector is obtained by inserting the DNA fragment shown in SEQ ID No.2 between the restriction enzyme cutting sites Kpn I and Sal I of the pMDC32 vector.

In the method of the third aspect, the reduction of the expression level and/or activity of GhHUB2 protein in the cotton recipient can be achieved by knocking out or inhibiting the expression of a gene encoding the GhHUB2 protein in the cotton recipient.

Further, this can be accomplished by any means that can accomplish this.

In the method of the second aspect and the method of the third aspect, the recombinant vector carrying the coding gene of the GhHUB2 protein or a gene editing tool used for knocking out or inhibiting expression of the coding gene of the GhHUB2 protein in the recipient cotton is introduced into the recipient cotton, and specifically may be: plant cells or tissues are transformed by conventional biological methods using Ti plasmids, Ri plasmids, plant viral vectors, direct DNA transformation, microinjection, conductance, agrobacterium mediation, etc., and the transformed plant tissues are grown into plants.

In the use of the first aspect, the method of the second aspect, and the method of the third aspect, the GhHUB2 protein may be a protein represented by any one of the following (a1) to (a 4):

(A1) protein with the amino acid sequence shown as 24 th-901 th positions of SEQ ID No. 1;

(A2) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the 24 th-901 th position of SEQ ID No.1 and has the same function;

(A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the amino acid sequence defined in any one of (A1) to (A2) and having the same function;

(A4) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).

Further, the fusion protein in (a4) is a GhHUB2 protein fused with 3 × FALG tag shown in SEQ ID No.1 as an amino acid sequence.

In the use according to the first aspect, the method according to the second aspect, and the method according to the third aspect, the gene encoding GhHUB2 protein is a DNA molecule as described in any one of (B1) to (B3):

(B1) the 70 th-2706 th site of SEQ ID No.2 or the DNA molecule shown in SEQ ID No. 2;

(B2) a DNA molecule which is hybridized with the DNA molecule defined by (B1) under strict conditions and encodes the GhHUB2 protein;

(B3) and (B) a DNA molecule which has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% of homology with the DNA sequence limited by (B1) or (B2) and encodes the GhHUB2 protein.

The stringent conditions may be hybridization with a solution of 6 XSSC, 0.5% SDS at 65 ℃ followed by washing the membrane once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

In the present invention, the cotton may be specifically a cotton institute 24 in cotton variety.

In the present invention, the fiber strength is embodied in particular as specific strength at break (cN/tex).

Experiments prove that: constructing an overexpression vector of the GhHUB2 gene, transforming cotton seed 24 in a receptor material by utilizing an agrobacterium-mediated genetic transformation method to obtain stably-inherited transgenic cotton, and detecting the quality of cotton fibers to obtain a novel cotton seed with increased fiber length and enhanced strength. Constructing a GhHUB2 gene interference vector, transforming cotton seed 24 in a receptor material by utilizing an agrobacterium-mediated genetic transformation method to obtain stably inherited transgenic cotton, and detecting the quality of cotton fibers to obtain a new cotton seed with reduced fiber length and reduced strength. The invention has important significance for cultivating new cotton varieties with better fiber quality.

Drawings

FIG. 1 is a schematic diagram of a cotton overexpression vector pMDC32-GhHUB2 vector.

FIG. 2 is T₀PCR identification of generation transgenic cotton, 402 is 17 transgenic cotton lines. CCRI 24 is cotton institute 24 (negative control) in the acceptor material, + pMDC32-GhHUB2 plasmid (positive control), M is DNA standard molecular weight. Since the GhHUB2 genome contains 18 introns and is 9519bp long, the target fragment cannot be detected by a negative control.

FIG. 3 shows the detection of T by qRT-PCR₀The expression level of GhHUB2 in transgenic cotton generation. CCRI 24 is cotton institute 24 in the acceptor material, 402 is GhHUB2 overexpression transgenic line. The experiment was repeated three times and the data are presented as mean ± SE.

FIG. 4 shows immunoblot assay T₀The GhHUB2 protein in transgenic cotton leaf is generated. CCRI 24 is cotton institute 24, 402-13, 402-20 and 402-47 in the receptor material is GhHUB2 overexpression transgenic line, and Marker is protein molecular weight standard.

FIG. 5 shows the detection of T by qRT-PCR₃The expression level of GhHUB2 in transgenic cotton generation. CCRI 24 is cotton seed 24, 402-13, 402-20 and 402-47 in acceptor material GhHUB2 superExpressing the transgenic line. Histone3 is used as an internal reference. The experiment was repeated three times and the data are presented as mean ± SE. Student's t-test, P<0.01。

FIG. 6 is T₃Mature fiber length of transgenic cotton and wild-type cotton. CCRI 24 is cotton institute 24, 402-13, 402-20 and 402-47 in the acceptor material is GhHUB2 overexpression transgenic line. A is T₃Mature fiber phenotype of generation transgenic cotton and wild type cotton, scale is 1 cm; b is T₃Statistics of mature fiber length, n-30, for transgenic and wild-type cotton generations, data expressed as mean ± SE, Student's t-test, × P<0.01。

FIG. 7 is T₃Fiber length of transgenic cotton and wild type cotton ovules cultured in vitro for 20 days. CCRI 24 is cotton institute 24, 402-13, 402-20 and 402-47 in the acceptor material is GhHUB2 overexpression transgenic line. A is the fiber phenotype of ovule culture with a scale of 1 cm; b is the statistics of the length of the cultured fiber of the ovule, n is more than or equal to 30, data are expressed as the mean value + -SE, Student' st-test,. P<0.01。

FIG. 8 is T₃Thickness of mature fiber cell wall of transgenic cotton generation. CCRI 24 is cotton institute 24, 402-13, 402-20 and 402-47 in the acceptor material is GhHUB2 overexpression transgenic line. A is T₃Microscopic observation of mature fiber cell wall paraffin sections of transgenic cotton and wild cotton, with a ruler of 20 μm; b is T₃Statistics of mature fiber cell wall thickness of generation transgenic cotton and wild type cotton, n.gtoreq.300, data expressed as mean. + -. SE, Student's t-test,. times.P<0.01。

FIG. 9 is a transmission electron microscope observation T₃Thickness of fiber cell wall 30 days after flowering of transgenic cotton generation. CCRI 24 is cotton institute 24, 402-13, 402-20 and 402-47 in the acceptor material is GhHUB2 overexpression transgenic line with scale of 2 μm.

FIG. 10 is a schematic diagram of the cotton interference vector pBI121-GhHUB2RNAi vector.

FIG. 11 is T₀The PCR identification of the generation transgenic cotton is that 401 is 13 transgenic cotton lines. CCRI 24 is cotton institute 24 (negative control) in the receptor material, + is pBI121-GhHUB2RNAi plasmid (positive control), M isDNA standard molecular weight. Since the wild-type genome does not contain the CaMV35S promoter, the target fragment was not detected by the negative control.

FIG. 12 is qRT-PCR detection of T₀The expression level of GhHUB2 in transgenic cotton generation. CCRI 24 is cotton institute 24 in acceptor material, 401 is GhHUB2 interference transgenic line. The experiment was repeated three times and the data are presented as mean ± SE.

FIG. 13 shows the detection of T by qRT-PCR₃The expression level of GhHUB2 in transgenic cotton generation. CCRI 24 is cotton institute 24, 401-34, 401-52 and 401-57 in the acceptor material is GhHUB2 interfering strain. Histone3 is used as an internal reference. The experiment was repeated three times and the data are presented as mean ± SE. Student's t-test, P<0.01。

FIG. 14 is T₃Mature fiber length of transgenic cotton and wild-type cotton. CCRI 24 is cotton institute 24, 401-34, 401-52 and 401-57 in the acceptor material is GhHUB2 interfering strain. A is T₃Mature fiber phenotype of generation transgenic cotton and wild type cotton, scale is 1 cm; b is T₃Statistics of mature fiber length, n-30, for transgenic and wild-type cotton generations, data expressed as mean ± SE, Student's t-test, × P<0.01。

FIG. 15 is T₃Fiber length of transgenic cotton and wild type cotton ovules cultured in vitro for 20 days. CCRI 24 is cotton institute 24, 401-34, 401-52 and 401-57 in the acceptor material is GhHUB2 interfering strain. A is the fiber phenotype of ovule culture with a scale of 1 cm; b is the statistics of the length of the cultured fiber of the ovule, n is more than or equal to 30, data are expressed as mean value + -SE, Student's t-test;. P<0.01。

FIG. 16 is T₃Thickness of mature fiber cell wall of transgenic cotton generation. CCRI 24 is cotton institute 24, 402-13, 401-34, 401-52 and 401-57 in the acceptor material is GhHUB2 interference strain. A is T₃Microscopic observation of mature fiber cell wall paraffin sections of transgenic cotton and wild cotton, with a ruler of 20 μm; b is T₃Statistics of mature fiber cell wall thickness of generation transgenic cotton and wild type cotton, n.gtoreq.300, data expressed as mean. + -. SE, Student's t-test,. times.P<0.01。

FIG. 17 is a transmission electron microscope observation T₃Thickness of fiber cell wall 30 days after flowering of transgenic cotton generation. CCRI 24 is cotton institute 24, 401-34, 401-52 and 401-57 in the acceptor material is GhHUB2 interfering strain with a scale of 2 μm.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

pMDC32 vector: is a product of The Arabidopsis Information Resource (TAIR), Stock #: CD3-738(http:// www.arabidopsis.org/servlets/TairObject.

pOSB209 vector: described in Ma et al, 2011, RMDAP a versatile, ready-to-usentolbox for multigene genetic transformation, publicly available from the Applicant and only available for use in the duplication of experiments.

Example 1 cultivation of transgenic Cotton with improved fiber Length and Strength and testing of fiber quality

First, obtaining transgenic Cotton with improved fiber Length and Strength

1. Cloning of GhHUB2 Gene

The nucleotide shown in SEQ ID No.2 was synthesized by Invitrogen, in which positions 1-69 were the 3 XFLAG tag sequence and positions 70-2706 were the sequence of the GhHUB2 gene. SEQ ID No.2 encodes the protein shown in SEQ ID No.1, the 1 st to 23 th positions of SEQ ID No.1 are 3 XFLAG labels, and the 24 th to 901 th positions are GhHUB2 protein sequences.

And (3) carrying out PCR amplification by using a synthesized DNA fragment shown in SEQ ID No.2 as a template and pMDC32-GhHUB2-F and pMDC32-GhHUB2-R as primers to obtain an amplification product.

pMDC32-GhHUB2-F：5’-GGTACC

ATGGAAAGCTTTGAATCTGAAG-3’；

pMDC32-GhHUB2-R：5’-GTCGACTCATATTTTCACAAACCGAACATCA-3’。

2. Construction of overexpression vectors

Carrying out double enzyme digestion on the amplification product obtained in the step 1 by Kpn I and Sal I, recovering the enzyme digestion product, and connecting the enzyme digestion product with a plant expression vector pMDC32 subjected to the same double enzyme digestion to obtain an over-expression vector pMDC32-GhHUB2 (figure 1).

3. Obtaining transgenic cotton

(1) Transformation of Agrobacterium

a. The preparation method of agrobacterium electric shock transformation competence comprises the following steps:

taking agrobacterium strain EHA105 to streak on YEP plate containing rifampicin, and culturing for 48h at 28 ℃ in an inverted mode until obvious bacterial colony is visible; picking a single colony in 5 mL YEP liquid culture medium containing rifampicin, shaking and culturing the single colony overnight at 250 rpm and 28 ℃ by a shaking table; inoculating 2 mL of overnight cultured bacterial liquid into 200 mL of YEP liquid culture medium containing Rif, shaking at 200rpm and 28 deg.C, and culturing to OD₆₀₀Up to 1.5 or more; carrying out ice-bath on the bacterial liquid for 10min, centrifuging at 4 ℃ and 4000 rpm for 10min, collecting thalli, and removing supernatant; resuspending the thallus with 150 mL of precooled Milli Q ultrapure water, centrifuging at 4000 rpm for 10min, collecting the thallus, and discarding the supernatant; resuspending the cells in 100 mL of precooled Milli Q ultrapure water, washing the cells for 2 times, and discarding the supernatant; resuspend the cells in 2 mL of pre-cooled 10% sterile glycerol, load into 1.5 mL centrifuge tubes, 100. mu.L per tube, snap-freeze with liquid nitrogen and store at-80 ℃ for future use.

b. The method for transforming agrobacterium by electric shock method is as follows:

thawing agrobacterium tumefaciens infected state in ice bath, adding about 50ng plasmid vector (pMDC32-GhHUB2), mixing gently, and ice-cooling for 15 min; adding the mixture into a precooled electric shock cup, and performing 1800V high-voltage electric shock; adding 600 μ L of precooled YEP into an electric shock cup, mixing uniformly, sucking out the bacterial liquid, shaking the bacterial liquid at 28 ℃ in a shaking table at 200rpm, and performing shaking culture for 1 h; sucking 40 mu L of bacterial liquid, uniformly coating the bacterial liquid on YEP plates (containing 50 mu g/mL Kan and 125 mu g/mL rifampicin) containing antibiotics by using an applicator, drying the surfaces of the plates, and carrying out inverted culture in a constant-temperature incubator at 28 ℃ for 48 hours until bacterial colonies are obviously visible; picking single colony, inoculating in YEB liquid culture medium (containing 50 ug/mL Kan and 125 ug/mL rifampicin), culturing at 28 deg.C and 220rpm with shaking overnight; taking 2 mu L of bacterial liquid as a template to perform bacterial liquid PCR so as to identify the positive clone of the agrobacterium. The primers used for identification were the 5' primer of GhHUB 2: 5'-ATGGAAAGCTTTGAATCTGAAGAAC-3' and the 3 ' primer of the GhHUB2 gene: 5'-TCATATTTTCACAAACCGAACATCA-3' (the theoretical amplification product is 70-2706 of SEQ ID No. 2), and the result shows that the vector pMDC32-GhHUB2 has been successfully transferred into Agrobacterium.

(2) Genetic transformation of cotton

a. Obtaining of explants

Seeds from cotton institute 24 (product of scientific trade company of cotton institute of Chinese academy of agricultural sciences) in the recipient material were dehulled. Sterilizing with 0.1% mercuric chloride for 5min, washing with sterile water for 3-5 times, sowing on sterile MS culture medium, and culturing under illumination for 5-7 d; cutting the hypocotyl into sections of about 0.5cm, and using the sections of the hypocotyl of the cotton aseptic seedlings of 5-7d as explants for agrobacterium infection transformation.

b. Agrobacterium mediated transformation and culture

The single colony of Agrobacterium containing the expression vector pMDC32-GhHUB2 verified above was picked and inoculated into liquid YEB medium (50. mu.g/ml Kan and 125. mu.g/ml rifampicin), and shake-cultured at 28 ℃ until OD₆₀₀Centrifuging at 5000rpm for 5min at a value of 0.5-0.7, resuspending with liquid MSB culture medium, co-culturing with hypocotyl dissection of 5-7D cotton aseptic seedling for 10min, sucking off explant surface bacterial liquid with aseptic filter paper, transferring into co-culture medium (MSB +0.1 mg/L2, 4-D +0.1mg/L KT +0.1mg/L IAA +30g/L glucose +2g/L Gelrite, pH 5.8) with a layer of filter paper laid on the surface, co-culturing at 21 deg.C in dark for 48h, transferring into resistance induction medium (MSB +0.1 mg/L2, 4-D +0.1mg/L KT +0.1mg/L IAA +500mg/L cephamycin +50mg/L hygromycin +30g/L glucose +2g/L Gelrite, pH 5.8), culturing for 3 weeks, transferring the induced callus with good growth medium (MSB +0.005 mg/L) into differentiation medium mg/L IAA +30g/L glucose +2g/L Gelrite, pH 5.8), transferring the embryoid into a seedling culture medium (MSB +0.1mg/L IBA +30g/L glucose +2g/L Gelrite, pH 5.8) to form a regenerated plant, and grafting the obtained transgenic regenerated plant onto a stock seedling by a cleft grafting method;taking a cotton seedling growing 4-5 true leaves in a nutrition pot as a stock, holding the seedling with the left hand, removing the top end part of the cotton seedling with a sharp scalpel with the right hand, only keeping 1-2 true leaves at the lower part, slightly splitting the top part from top to bottom by about 1cm, inserting a regenerated seedling into the split part, winding a joint part with a thread, sealing the watered nutrition pot and the newly-grafted regenerated plant with a plastic bag, and placing the nutrition pot and the newly-grafted regenerated plant under the condition of shading light at 30-36 ℃ for growth; and 7-10d, removing the plastic bag, untying the plastic rope, performing shading culture for 7-10d, and then placing the nutrition pot into a flowerpot for field planting.

(3) Detection of transgenic Cotton

a. Hygromycin detection

When 2-3 true leaves grow from the transformed single seedling, the hygromycin leaf smearing primary screening experiment is carried out on the transformed single seedling. And (3) coating hygromycin with the concentration of 80mg/L on the transformed single plants, observing the color of the coated part after coating for 7-10 days, eliminating the single plants with yellow coated leaves, then carrying out second-round screening, continuously coating for 3 times, and selecting the single plants with the coated leaves still being green. Marking the hygromycin resistant single plant according to the last screening result, and sampling for indoor molecular biological detection.

b. DNA level detection

And (3) PCR detection: the transgenic cotton genome DNA is used as a template, the plasmid pMDC32-GhHUB2 is used as a positive control, the genome DNA of untransformed cotton is used as a negative control, a transgenic plant is identified by PCR amplification, and the 5' primer of the GhHUB2 gene is used: 5'-ATGGAAAGCTTTGAATCTGAAGAAC-3' and the 3 ' primer of the GhHUB2 gene: 5'-TCATATTTTCACAAACCGAACATCA-3', the theoretical amplification product is the 70-2706 site of SEQ ID No.2, and the reaction system is shown in Table 1:

TABLE 1 reaction System for PCR detection

The PCR reaction program is: a first round: denaturation at 94 deg.C for 5 min; and a second round: denaturation at 94 ℃ for 30sec, annealing at 55 ℃ for 30sec, extension at 72 ℃ for 2min, 35 cycles; and a third round: extension at 72 ℃ for 10 min. After the reaction, the result was detected by electrophoresis on 1.0% agarose gel.

Selecting the plants capable of amplifying specific GhHUB2 gene fragments (2637bp, 70-2706 of SEQ ID No. 2) as PCR positive plants, and detecting to obtain 17 positive transgenic cotton strains (figure 2). Since the GhHUB2 genome contains 18 introns and is 9519bp long, the target fragment cannot be detected by a negative control.

c. RNA level detection

qRT-PCR detection: and (c) further performing qRT-PCR detection to detect whether the GhHUB2 gene in the PCR positive plant in the step (b) is transcribed and the expression level of the GhHUB2 gene in the positive plant. Extracting total RNA of the PCR positive plants in the step b by an SDS method, taking RNA with good integrity and no pollution as a template, and carrying out reverse transcription on the RNA by using M-MLV enzyme (product number M1701, Promega corporation) to obtain cDNA, wherein a reverse transcription reaction system is shown in Table 2:

TABLE 2 reverse transcription reaction System

Mix gently, denature at 70 ℃ for 5min, then immediately ice-wash.

The following system (table 3) was prepared in advance, mixed well, and added to the reaction solution:

TABLE 3 formulation system

Mix gently and react at 42 ℃ for 1 h. The reaction was then diluted 4-fold with distilled water to obtain a cDNA solution, which was stored at-20 ℃ until use.

The cotton histone H3 gene GhHIS3 is selected as an internal standard for qRT-PCR quantification, and the used primers are GhHIS3-RT forward primers: 5'-GGCATACCTTGTGGGTCTTTTTGA-3' and GhHIS3-RT reverse primer: 5'-CTACCACTACCATCATGGC-3' are provided. The primer used by the target gene GhHUB2 is a GhHUB2-RT forward primer: 5'-GATGAGTTCCAAATGAAGTTGG-3' and GhHUB2-RT reverse primer: 5'-GTCAAAACACACACCACATTTGAG-3' are provided. Detection was carried out using fluorescent quantitative premixed dye SYBR Premix Ex Taq (cat No. RR420A, Takara Co.) by Takara in a fluorescent quantitative PCR instrument (model CFX96, Bio-Rad Co., U.S.A.).

The PCR reaction program is: a first round: denaturation at 95 ℃ for 30 sec; and a second round: denaturation at 95 ℃ for 10sec, annealing at 57 ℃ for 30sec, Read plate, 40 cycles; and a third round: pre-denaturation at 95 ℃ for 10sec, 65 ℃ to 95 ℃ and reading of the dissolution curve by the Read plate.

The results show that the overexpression of the GhHUB2 enables the expression level of the GhHUB2 to be remarkably increased compared with the wild type, wherein the GhHUB2 overexpression strains 402-13, 402-20 and 402-47 are most remarkably upregulated, and the expression level of the GhHUB2 is 14-15 times upregulated relative to the wild type (figure 3).

d. Protein level detection

Immunoblotting experiments: and (c) further carrying out immunoblot detection to detect whether the GhHUB2 gene is expressed in the positive transgenic plant with the most significant expression level up-regulated in the step c. Extracting total protein of a transgenic plant, carrying out SDS-PAGE gel electrophoresis separation, transferring the protein to an acetate fiber membrane by using an electrotransfer device for immunoblotting hybridization, sealing, adding a specific antibody FLAG-Tag Mouse mAb (the dilution ratio is 1: 5000, the product number is # F3165, purchased from Sigma-Aldrich company), reacting with a secondary antibody, adding alkali horseradish peroxidase-labeled goat anti-Mouse IgG (the dilution ratio is 1: 5000, the product number is 5220-0341, purchased from KPL company) diluted by using sealing liquid, washing the hybridization membrane for 3 times, placing the nitrocellulose membrane into a buffer solution containing a chromogenic substrate under a light-proof condition, and detecting a chromogenic signal by using a full-automatic chemiluminescence imaging analysis system (model Tanon-5200, Shanghai-Tian-energy technology Limited company). The specific band (figure 4) with about 100kDa can be developed as a positive plant of the immunoblot, indicating that GhHUB2 is successfully expressed in the transgenic cotton.

e、T₃Expression level of GhHUB2 in transgenic cotton generation

The 3 overexpression strains 402-13, 402-20 and 402-47 with the most significant expression quantity of GhHUB2 are self-pollinated to obtain T₃And generating transgenic cotton material. And detecting T according to the method of c₃Expression level of transgenic cotton GhHUB2 (FIG. 5). The result shows that the overexpression of the GhHUB2 can improve the table of GhHUB2 in transgenic cotton progenyAnd (4) obtaining the amount.

Second, detection of cotton fiber quality

1、T₃Detection of fiber length of mature cotton fiber

Manual picking T₃Cotton bolls in the middle of the plants, mature at the same period as cotton 24 in the GhHUB2 transgenic cotton and control cotton, were measured by cotton seed combing. Straightening the fiber along the middle abdominal furrow of the cottonseed, and combing the fiber by using a comb. And then, downwards sticking the cotton seed bellies on a black wool board, and measuring by using a steel ruler to obtain the length of the cotton fibers. The results show (FIG. 6) that the average length of the wild type fibers is 28.1 mm; the average lengths of fibers of GhHUB2 overexpression lines 402-13, 402-20 and 402-47 respectively reach 30.3mm, 30.6mm and 30.2 mm. The fibers of the over-expressed strain were increased by about 7.49% -8.99% relative to wild type.

2. In vitro culture of ovules to detect fiber length

At 8-9 am, naturally grown T was taken₃Cotton bolls in the middle of plants on the day of flowering of the transgenic cotton and the wild type are replaced and stored at low temperature for later use; soaking cotton boll in 75% ethanol, sterilizing for 2min, and shaking continuously; transferring sterilized cotton boll into sterile water, cleaning for 2min, and shaking continuously; repeatedly cleaning for 3 times; removing the top of the cotton boll by using a scalpel, longitudinally cutting the ovary wall from the top end, peeling off the cotton boll, carefully taking out the ovule by using a pair of tweezers, and lightly placing the ovule in the BT culture medium to enable the ovule to be suspended on the surface of the culture medium; and (5) culturing in a constant temperature incubator at 30 ℃ for 20 days in the dark, photographing and counting the fiber length. The results show (FIG. 7) that the average length of the wild type cotton fiber cultured in vitro was 17.9 mm; the average length of GhHUB2 super-expression material fiber is 20.2mm, 20.5mm and 20.1mm respectively. The result of in vitro ovule culture is consistent with the field test, and the GhHUB2 overexpression material fiber is obviously increased.

3、T₃Fiber observation of cell wall thickness of mature cotton fiber

Selection of T of Simultaneous maturation₃The cotton bolls in the middle of transgenic cotton and wild plants are replaced, and fibers in the same part are made into cotton fiber cross-cut paraffin sections with the thickness of 8 mu m. Observations were made under a microscope and cell wall thickness was counted. The results show (FIG. 8) that wild-type cotton fibersThe cell wall thickness is 2.19 μm; the cell wall thickness of the over-expression GhHUB2 fiber is 2.88-3.12 μm, which is obviously increased compared with the wild cotton fiber.

4. Transmission electron microscope observation T₃Cell wall thickness of cotton fiber

Selection of T₃The transgenic cotton and the boll 30 days after the middle flowering of the wild type plant were used as generations, and the fiber of the same portion was cut into an ultrathin section with a thickness of 50nm and observed under a transmission electron microscope (model JEM-1400, JEOL, Japan). The results show (FIG. 9) that the fibrous cell wall of the GhHUB2 overexpression line is significantly thicker than the wild type.

5、T₅Detection of cotton fiber quality

Manual picking T₅Cotton bolls matured at the same time period as cotton plant 24 in the GhHUB2 generation transgenic cotton were subjected to machine ginning. The weight of each fiber sample was around 10 g. The average length of the upper half (mm), the strength at break (cN/tex), the uniformity and the micronaire coefficient were each determined according to HVICC standard using a high-capacity cotton fiber tester (model HFT9000, Premier, India). The determination is completed by the cotton quality supervision and inspection test center of the Ministry of agriculture of Henan, an Yang.

The results are shown in Table 4.

TABLE 4 comparison of GhHUB2 transgenic Cotton fiber traits with wild-type Cotton

Sample (I)	Fiber length (mm)	Fiber Strength (cN/tex)	Fiber uniformity (%)	Micronaire value
					CCRI24	29.44±0.49	31.73±0.82	85.47±0.96	4.24±0.18
402-13	31.69±0.62**	33.79±0.87**	85.37±1.01	4.92±0.21**
					402-20	31.97±0.74**	33.92±0.91**	85.31±1.15	4.97±0.23**
402-47	31.62±0.51**	33.35±0.74*	85.41±1.06	4.83±0.16**

As can be seen from the fiber quality test results (Table 4), the over-expression of GhHUB2 significantly increased the length of cotton fibers, resulting in a 7.40% -8.59% increase in transgenic cotton fibers over control cotton 24. Meanwhile, the strength of the transgenic cotton fiber is improved by 5.10 to 6.90 percent compared with the strength of the cotton fiber 24 in the contrast.

The results show that the method obtains the new transgenic cotton germplasm with increased fiber length and enhanced strength by over-expressing the GhHUB2 gene in cotton.

Example 2 cultivation of transgenic Cotton with reduced fiber Length and Strength and testing of fiber quality

One, obtaining transgenic Cotton with reduced fiber Length and Strength

1. Cloning of GhHUB2 gene interference fragment

The 2491-position 2705 of the SEQ ID No.2 is amplified by PCR as an interference fragment by taking the synthesized DNA fragment shown in the SEQ ID No.2 as a template and pBI121-GhHUB2-F and pBI121-GhHUB2-R as primers to obtain an amplification product.

pBI121-GhHUB2-F：5’-GGGGACAAGTTTGTACAAAAAAGCAGTACTGCAATACAGAAGCTTCAGGATGA-3' (the underlined part is the attB1 sequence for subsequent recombination reactions);

pBI121-GhHUB2-R：5’-GGGGACCACTTTGTACAAGAAAGCTGGGTCATATTTTCACAAACCGAACATC-3' (the underlined part is the attB2 sequence for subsequent recombination reactions).

2. Construction of interference vectors

The amplification product obtained in step 1 was subjected to recombination reaction with pOSB209 vector by Gateway BP clone II Enzymemix (ThermoFisher Co., Ltd., product No. 11789020). Reaction system 10 μ L:

TABLE 5 recombination reaction System

The reaction was carried out at 25 ℃ for 12h, E.coli TOP10 was transformed to identify positive clones, and vector plasmids were extracted. The obtained vector was subjected to double digestion with Asc I and I-Sce I, and the digested product was recovered and ligated to pBI121 vector subjected to the same double digestion to obtain interference vector pBI121-GhHUB2RNAi (FIG. 10).

The structure of the interference vector pBI121-GhHUB2RNAi is described as follows: a small fragment between the enzyme cutting sites Asc I and I-Sce I of the pBI121 vector is replaced by a recombinant plasmid of a DNA fragment shown in SEQ ID No. 3. The position 134-1479 of SEQ ID No.3 is the CaMV35S promoter, the position 1589-1803 is the GhHUB2-RNAi forward sequence, the position 18014-3602 is the PDK intron, the position 3703-3917 is the GhHUB2-RNAi reverse sequence, and the position 4048-4813 is the OCS terminator.

3. Obtaining transgenic cotton

(1) Transformation of Agrobacterium

As described in example 1. The agrobacterium identification primer is as follows:

CaMV35S-F：5’-GATGCAGTCAAAAGATTCAGGAC-3’；

GhHUB2-R：5’-CATATTTTCACAAACCGAACATCATT-3’。

the theoretical amplification product is 757-1803 th site of SEQ ID No.3, and the result shows that the vector pBI121-GhHUB2RNAi has been successfully transferred into Agrobacterium.

(2) Genetic transformation of cotton

As described in example 1. 50mg/L kanamycin was used instead of hygromycin in the cotton resistant callus induction medium.

(3) Detection of transgenic Cotton

a. Kanamycin detection

When 2-3 true leaves grow from the transformed single plant seedling, a kanamycin leaf smearing primary screening experiment is carried out on the transformed single plant seedling. And (3) coating the kanamycin with the concentration of 80mg/L on the transformed single plant, observing the color of a coating part after coating for 7-10 days, eliminating the single plant with yellow leaf coating points, then carrying out second-round screening, continuously coating for 3 times in such a way, and selecting the single plant with the coated leaf which is still green. And marking the kanamycin-resistant individual plant according to the last screening result, and sampling for indoor molecular biological detection.

b. DNA level detection

And (3) PCR detection: the transgenic cotton genome DNA is used as a template, plasmid pBI121-GhHUB2RNAi is used as a positive control, the genome DNA of untransformed cotton is used as a negative control, a transgenic plant is identified by PCR amplification, and a primer 5' of a CaMV35S promoter: 5'-GATGCAGTCAAAAGATTCAGGAC-3' and the 3 ' primer of the GhHUB2 gene: 5'-CATATTTTCACAAACCGAACATCATT-3', the theoretical amplification product is 757-1803 site of SEQ ID No.3, and the reaction system is shown in Table 6:

TABLE 6 reaction System for PCR detection

The PCR reaction program is: a first round: denaturation at 94 deg.C for 5 min; and a second round: denaturation at 94 ℃ for 30sec, annealing at 55 ℃ for 30sec, extension at 72 ℃ for 1min, 35 cycles; and a third round: extension at 72 ℃ for 10 min. After the reaction, the result was detected by electrophoresis on 1.0% agarose gel.

Selecting the plants capable of amplifying the specific GhHUB2 gene fragment (1047bp, 757-position 1803 of SEQ ID No. 3) as PCR positive plants, and detecting to obtain 13 positive transgenic cotton lines (FIG. 11). While the non-transgenic wild-type plant (CCRI 24) did not detect the band of interest (since the wild-type plant did not contain the CaMV35S promoter, the negative control did not detect the fragment of interest).

c. RNA level detection

qRT-PCR detection: in order to detect the expression level of the GhHUB2 gene in the PCR positive GhHUB2 interference plants in the step b, qRT-PCR detection is further carried out. The procedure is as described in example 1.

The results show that interference with GhHUB2 significantly reduces the expression level of GhHUB2 compared with the wild type, wherein the GhHUB2 interference strain lines 401-34, 401-52 and 401-57 are reduced most significantly, and the expression level of GhHUB2 is reduced by more than 85% compared with the wild type (FIG. 12).

d、T₃Expression level of GhHUB2 in transgenic cotton generation

The 3 interference strains 401-34, 401-52 and 401-57 with the most significant reduction of the expression level of GhHUB2 are self-pollinated to obtain T₃And generating transgenic cotton material. And detecting T according to the method of c₃Expression level of transgenic cotton GhHUB2 (FIG. 13). The result shows that the interference with the GhHUB2 can reduce the expression level of GhHUB2 in transgenic cotton progeny.

Second, detection of cotton fiber quality

1、T₃Detection of fiber length of mature cotton fiber

Manual picking T₃Cotton bolls in the middle of the plants, mature at the same period as cotton 24 in the GhHUB2 transgenic cotton and control cotton, were measured by cotton seed combing. Straightening the fiber along the middle abdominal furrow of the cottonseed, and combing the fiber by using a comb. Then, the cotton seed bellies are attached to the black wool board downwards, and the cotton seed bellies are measured by a steel ruler to obtain cotton fibersThe dimension length. The results show (figure 14) that the average length of wild type fibres is 28.1 mm; the average length of fibers of GhHUB2 interference strains 401-34, 401-52 and 401-57 reaches 26.4mm, 25.7mm and 26.2mm respectively. The fiber length of the interfering strain is reduced by about 5.85-8.24% relative to the wild type.

2. In vitro culture of ovules to detect fiber length

At 8-9 am, naturally grown T was taken₃Cotton bolls in the middle of plants on the day of flowering of the transgenic cotton and the wild type are replaced and stored at low temperature for later use; soaking cotton boll in 75% ethanol, sterilizing for 2min, and shaking continuously; transferring sterilized cotton boll into sterile water, cleaning for 2min, and shaking continuously; repeatedly cleaning for 3 times; removing the top of the cotton boll by using a scalpel, longitudinally cutting the ovary wall from the top end, peeling off the cotton boll, carefully taking out the ovule by using a pair of tweezers, and lightly placing the ovule in the BT culture medium to enable the ovule to be suspended on the surface of the culture medium; and (5) culturing in a constant temperature incubator at 30 ℃ for 20 days in the dark, photographing and counting the fiber length. The results show (FIG. 15) that the average length of the wild type cotton fiber cultured in vitro was 17.9 mm; the average length of the interference material fiber of the GhHUB2 is 16.2mm, 16.0mm and 16.4mm respectively. The results of in vitro ovule culture were consistent with field trials with GhHUB2 interfering with a significant reduction in material fiber length.

3、T₃Fiber observation of cell wall thickness of mature cotton fiber

Selection of T of Simultaneous maturation₃The cotton bolls in the middle of transgenic cotton and wild plants are replaced, and fibers in the same part are made into cotton fiber cross-cut paraffin sections with the thickness of 8 mu m. Observations were made under a microscope and cell wall thickness was counted. The results show (FIG. 16) that the wild type cotton fiber cell wall thickness was 2.19 μm; the interfering GhHUB2 fiber has a cell wall thickness of 1.66-1.71 μm, which is obviously reduced compared with wild cotton fiber.

Selection of T₃The transgenic cotton and the boll 30 days after the middle flowering of the wild type plant were used as generations, and the fiber of the same portion was cut into an ultrathin section with a thickness of 50nm and observed under a transmission electron microscope (model JEM-1400, JEOL, Japan). The results show (FIG. 17) GhHUB2 interfered with a significant reduction in fiber cell wall thickness of the strain compared to wild type.

5、T₅Detection of cotton fiber quality

Manual picking T₅Cotton bolls matured at the same time period as cotton plant 24 in the GhHUB2 generation transgenic cotton were subjected to machine ginning. The weight of each fiber sample was around 10 g. The average length of the upper half (mm), the strength at break (cN/tex), the uniformity and the micronaire coefficient were each determined according to HVICC standard using a high-capacity cotton fiber tester (model HFT9000, Premier, India). The determination is completed by the cotton quality supervision and inspection test center of the Ministry of agriculture of Henan, an Yang. The results are shown in Table 7.

TABLE 7 comparison of GhHUB2 transgenic Cotton fiber traits with wild-type Cotton

Sample (I)	Fiber length (mm)	Fiber Strength (cN/tex)	Fiber uniformity (%)	Micronaire value
					CCRI24	29.44±0.49	31.73±0.82	85.47±0.96	4.24±0.18
401-34	27.75±0.36**	29.37±0.76**	86.14±0.83	3.46±0.16**
					401-52	27.37±0.48**	29.14±0.88**	85.48±0.94	3.35±0.17**
401-57	27.63±0.44**	29.56±0.81*	85.51±1.07	3.60±0.17**

As can be seen from the fiber quality test results (table 7), interference with GhHUB2 significantly reduced the length of cotton fibers, resulting in a 5.74% -7.03% reduction in transgenic cotton fibers relative to control cotton plant 24. Meanwhile, the strength of the transgenic cotton fiber is also reduced by 6.42-7.03 percent compared with that of the cotton 24 in the contrast.

The results show that the method obtains the new transgenic cotton germplasm with reduced fiber length and strength by interfering the GhHUB2 gene in cotton.

<110> university of agriculture in China

Application of <120> GhHUB2 protein in regulation and control of length and strength of cotton fiber

<130>GNCLN180958

<160>3

<170>PatentIn version 3.5

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tctctctgcc gacagtggtc ccaaagatgg acccccaccc acgaggagca tcgtggaaaa 1320

agaagacgtt ccaaccacgt cttcaaagca agtggattga tgtgacatct ccactgacgt 1380

aagggatgac gcacaatccc actatccttc gcaagaccct tcctctatat aaggaagttc 1440

atttcatttg gagaggacac gctcgaggct agcatggatc tcgggcccca aataatgatt 1500

ttattttgac tgatagtgac ctgttcgttg caacaaattg atgagcaatg cttttttata 1560

atgccaactt tgtacaaaaa agcaggctac tgcaatacag aagcttcagg atgagattaa 1620

aaattgcaag aatattctca aatgtggtgt gtgttttgac cggccaaaag aggtagtaat 1680

tgttaagtgc tatcacctat tttgcaatcc atgtatacag agaaatttag agatacggca 1740

tcgaaagtgc cccggctgtg gaactgcatt tggtcagaat gatgttcggt ttgtgaaaat 1800

atgacccagc tttcttgtac aaagttggca ttataagaaa gcattgctta tcaatttgtt 1860

gcaacgaaca ggtcactatc agtcaaaata aaatcattat ttgccatcca gctgcagctc 1920

ctcgaggaat tcggtacccc aattggtaag gaaataatta ttttcttttt tccttttagt 1980

ataaaatagt taagtgatgt taattagtat gattataata atatagttgt tataattgtg 2040

aaaaaataat ttataaatat attgtttaca taaacaacat agtaatgtaa aaaaatatga 2100

caagtgatgt gtaagacgaa gaagataaaa gttgagagta agtatattat ttttaatgaa 2160

tttgatcgaa catgtaagat gatatacggc cggtaagagg ttccaacttt caccataatg 2220

aaataagatc actaccgggc gtattttttg agttatcgag attttcagga gctaaggaag 2280

ctaaaatgga gaaaaaaatc actggatata ccaccgttga tatatcccaa tggcatcgta 2340

aagaacattt tgaggcattt cagtcagttg ctcaatgtac ctataaccag accgttcagc 2400

tggatattac ggccttttta aagaccgtaa agaaaaataa gcacaagttt tatccggcct 2460

ttattcacat tcttgcccgc ctgatgaatg ctcatccgga attccgtatg gcaatgaaag 2520

acggtgagct ggtgatatgg gatagtgttc acccttgtta caccgttttc catgagcaaa 2580

ctgaaacgtt ttcatcgctc tggagtgaat accacgacga tttccggcag tttctacaca 2640

tatattcgca agatgtggcg tgttacggtg aaaacctggc ctatttccct aaagggttta 2700

ttgagaatat gtttttcgtc tcagccaatc cctgggtgag tttcaccagt tttgatttaa 2760

acgtggccaa tatggacaac ttcttcgccc ccgttttcac catgggcaaa tattatacgc 2820

aaggcgacaa ggtgctgatg ccgctggcga ttcaggttca tcatgccgtc tgtgatggct 2880

tccatgtcgg cagaatgctt aatgaattac aacagtactg cgatgagtgg cagggcgggg 2940

cgtaatcgcg tggatccggc ttactaaaag ccagataaca gtatgcgtat ttgcgcgctg 3000

atttttgcgg tataagaata tatactgata tgtcgggccc ataatagtaa ttctagctgg 3060

tttgatgaat taaatatcaa tgataaaata ctatagtaaa aataagaata aataaattaa 3120

aataatattt ttttatgatt aatagtttat tatataatta aatatctata ccattactaa 3180

atattttagt ttaaaagtta ataaatattt tgttagaaat tccaatctgc ttgtaattta 3240

tcaataaaca aaatattaaa taacaagcta aagtaacaaa taatatcaaa ctaatagaaa 3300

cagtaatcta atgtaacaaa acataatcta atgctaatat aacaaagcgc aagatctatc 3360

attttatata gtattatttt caatcaacat tcttattaat ttctaaataa tacttgtagt 3420

tttattaact tctaaatgga ttgactatta attaaatgaa ttagtcgaac atgaataaac 3480

aaggtaacat gatagatcat gtcattgtgt tatcattgat cttacatttg gattgattac 3540

agttgggaaa ttgggttcga aatcgataag cttggatcct ctagagagct gcagctggat 3600

ggcaaataat gattttattt tgactgatag tgacctgttc gttgcaacaa attgataagc 3660

aatgctttct tataatgcca actttgtaca agaaagctgg gtcatatttt cacaaaccga 3720

acatcattct gaccaaatgc agttccacag ccggggcact ttcgatgccg tatctctaaa 3780

tttctctgta tacatggatt gcaaaatagg tgatagcact taacaattac tacctctttt 3840

ggccggtcaa aacacacacc acatttgaga atattcttgc aatttttaat ctcatcctga 3900

agcttctgta ttgcagtagc ctgctttttt gtacaaagtt ggcattataa aaaagcattg 3960

ctcatcaatt tgttgcaacg aacaggtcac tatcagtcaa aataaaatca ttatttgggg 4020

cccgagatcc atgctagctc tagagtcctg ctttaatgag atatgcgaga cgcctatgat 4080

cgcatgatat ttgctttcaa ttctgttgtg cacgttgtaa aaaacctgag catgtgtagc 4140

tcagatcctt accgccggtt tcggttcatt ctaatgaata tatcacccgt tactatcgta 4200

tttttatgaa taatattctc cgttcaattt actgattgta ccctactact tatatgtaca 4260

atattaaaat gaaaacaata tattgtgctg aataggttta tagcgacatc tatgatagag 4320

cgccacaata acaaacaatt gcgttttatt attacaaatc caattttaaa aaaagcggca 4380

gaaccggtca aacctaaaag actgattaca taaatcttat tcaaatttca aaaggcccca 4440

ggggctagta tctacgacac accgagcggc gaactaataa cgttcactga agggaactcc 4500

ggttccccgc cggcgcgcat gggtgagatt ccttgaagtt gagtattggc cgtccgctct 4560

accgaaagtt acgggcacca ttcaacccgg tccagcacgg cggccgggta accgacttgc 4620

tgccccgaga attatgcagc atttttttgg tgtatgtggg ccccaaatga agtgcaggtc 4680

aaaccttgac agtgacgaca aatcgttggg cgggtccagg gcgaattttg cgacaacatg 4740

tcgaggctca gcaggacctg caggcatgca agctagctta ctagtgatgc atattctata 4800

gtgtcaccta aatctgcggc cgctctagaa ctagtggatc ccccgggctg caggaattcg 4860

atatcaagct tatcgatacc gtcgacctcg agggggggcc cggtacccaa ttcgccctat 4920

agtgagtcgt attaattaag tttaaac 4947

Claims

The application of GhHUB2 protein or its coding gene in any one of the following:

(a1) regulating and controlling the length of cotton fibers;

(a2) regulating and controlling the strength of cotton fibers;

(a3) regulating the cell wall thickness of cotton fibers;

(a4) regulating and controlling the micronaire value of the cotton fibers;

the GhHUB2 protein is a protein with an amino acid sequence shown as 24 th to 901 th positions of SEQ ID No. 1.
2. Use according to claim 1, characterized in that: the higher the expression level and/or activity of the GhHUB2 protein or the coding gene thereof in the cotton, the longer the fiber length of the cotton, the stronger the fiber strength, the thicker the fiber cell wall thickness and/or the higher the micronaire value of the fiber; the lower the expression level and/or activity of the GhHUB2 protein or the coding gene thereof in the cotton, the shorter the fiber length of the cotton, the weaker the fiber strength, the thinner the fiber cell wall thickness and/or the lower the micronaire value of the fiber.
3. Use according to claim 1 or 2, characterized in that: the coding gene of the GhHUB2 protein is a DNA molecule shown in 70 th-2706 th positions of SEQ ID No. 2.
4. A method for breeding cotton having at least one of the traits shown in (b1) - (b4), comprising the step of increasing the expression amount and/or activity of GhHUB2 protein in recipient cotton;

(b1) the length of the fiber is increased;

(b2) the strength of the fiber is enhanced;

(b3) thickening the thickness of the fiber cell wall;

(b4) the micronaire value of the fibres increases;

the GhHUB2 protein is a protein with an amino acid sequence shown as 24 th to 901 th positions of SEQ ID No. 1.
5. A method for breeding cotton having at least one of the traits (c1) - (c4) below, comprising the step of decreasing the expression level and/or activity of GhHUB2 protein in recipient cotton;

(c1) the fiber length is shortened;

(c2) the strength of the fibers is weakened;

(c3) the thickness of the fiber cell wall is reduced;

(c4) a decrease in the micronaire value of the fibre;

the GhHUB2 protein is a protein with an amino acid sequence shown as 24 th to 901 th positions of SEQ ID No. 1.
6. The method of claim 4, wherein: the expression level and/or activity of GhHUB2 protein in acceptor cotton is increased by introducing the coding gene of GhHUB2 protein into the acceptor cotton.
7. The method of claim 6, wherein: the coding gene of the GhHUB2 protein is introduced into the recipient cotton in the form of a recombinant vector.
8. The method of claim 5, wherein: the reduction of the expression quantity and/or activity of GhHUB2 protein in receptor cotton is realized by knocking out or inhibiting expression of a coding gene of the GhHUB2 protein in the receptor cotton.
9. The method according to any one of claims 4-8, wherein: the coding gene of the GhHUB2 protein is a DNA molecule shown in 70 th-2706 th sites of SEQ ID No. 2.