CN111662921A - Cultivation method and application of transgenic cotton tag strain tracing positive end of microtubule in cotton cell - Google Patents

Abstract

The invention discloses a cultivation method and application of a transgenic cotton tag strain for tracing the positive end of a microtubule in a cotton cell. The method comprises the following steps: introducing the encoding gene of the fusion protein into receptor cotton to obtain transgenic cotton; the fusion protein is formed by fusing a microtubule positive terminal binding protein EB1b and a fluorescent protein mCherry. The laser confocal imaging system can clearly observe the dynamic change of tubulin in the living cells of the transgenic cotton in the life activity process of the cotton, and the obtained transgenic cotton does not influence the growth and development of the cotton, the length of cotton fibers and the yield of the cotton and can be used as a standard strain in the cotton production and research industry. The transgenic cotton obtained by the invention can be used for research on cotton growth and development, cell division, organelle transportation and the like, particularly can be used for microtubule dynamic analysis in the cotton fiber development process, and has important application value in the mechanism research aspect of cotton fiber quality improvement.

Description

Cultivation method and application of transgenic cotton tag strain tracing positive end of microtubule in cotton cell

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a cultivation method and application of a transgenic cotton tag strain for tracing a positive end of a microtubule in a cotton cell.

Background

Cotton (Gossypium) is one of the world's important commercial crops and is also the leading natural fiber source of the textile industry worldwide. The main product of cotton is cotton fiber, and the properties of cotton fiber directly determine the quality of cotton. The quality of cotton fiber includes cotton fiber length, cotton fiber fineness, cotton fiber strength, etc., which are closely related to cotton fiber cell development.

The cotton fiber cell is a single cell formed by the specific differentiation and development of the ovule exonucellus epidermal cell, and is an ideal material for researching the polar growth of the single cell and the cellulose synthesis. There are two types of cytoskeleton in plant cells: microtubules and microwires. Research shows that the microtubule skeleton is closely related to the determination of the growth direction of plant cells and cellulose synthesis, so that the research on the structure and dynamic characteristics of microtubules in cotton fiber cells is of great significance for analyzing the growth mechanism of cotton fiber cells and the genetic improvement of cotton fibers.

The major component of microtubules is tubulin, which mainly includes alpha-tubulin, beta-tubulin and microtubule-binding protein. In plant cells, microtubules are constantly changing. The subunits at the two ends of the tubulin are continuously polymerized and depolymerized, and the end with the polymerization speed higher than the depolymerization speed is the positive end of the microtubule.

Disclosure of Invention

The invention utilizes fluorescent protein to mark the positive end of a growing microtubule in a cotton cell, and obtains transgenic cotton which can observe the microtubule dynamics in the growth process of living cotton fiber cells. All tissues of the transgenic cotton, such as leaf epidermal cells, cotton fiber cells and the like, have the expression of mCherry-EB1b fusion protein, and the dynamic change of microtubules in living cells in the growth and development process of cotton can be clearly observed by applying a laser confocal imaging system.

The invention firstly protects a method for cultivating a transgenic cotton microtubule tag strain.

The method for cultivating the transgenic cotton microtubule tag strain comprises the following steps: introducing the encoding gene of the fusion protein into receptor cotton to obtain transgenic cotton;

the fusion protein is formed by fusing microtubule positive terminal binding protein EB1b and fluorescent protein.

In the method, the microtubule positive terminal binding protein EB1b (the amino acid sequence is shown in the 241-529 th site of the sequence 2) can be bound to the positive terminal of the growing microtubule, and the dynamic change of the microtubule in the cotton cell can be observed by fusion expression of the microtubule positive terminal binding protein EB1b and the fluorescent protein in the cotton cell.

In the above method, the fluorescent protein may be a common fluorescent protein in the prior art, and in a specific embodiment of the present invention, the fluorescent protein is a red fluorescent protein mCherry, and the fusion protein is formed by fusing a microtubule positive-terminal binding protein EB1b and the red fluorescent protein mCherry.

Further, the fusion protein is a protein shown in a) or b) or c) or d) as follows:

a) the amino acid sequence is a protein shown in a sequence 2;

b) a fusion protein obtained by connecting a label to the N end and/or the C end of the protein shown in the sequence 2;

c) the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 2;

d) and (b) a protein having a homology of 75% or more than 75% with the amino acid sequence shown in the sequence 2 and having the same function.

In order to facilitate the purification of the protein in a), the amino terminal or the carboxyl terminal of the protein shown in the sequence 2 in the sequence table can be connected with a label shown in the table 1.

TABLE 1 sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The protein of c) above, wherein the substitution and/or deletion and/or addition of one or more amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.

The protein in the c) can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

The gene encoding the protein of c) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in sequence No. 1, and/or performing missense mutation of one or several base pairs, and/or connecting the coding sequence of the tag shown in Table 1 to the 5 'end and/or 3' end thereof.

In the above d), "homology" includes an amino acid sequence having 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more homology with the amino acid sequence represented by the sequence 2 of the present invention.

Further, the encoding gene of the fusion protein is a gene shown in the following 1) or 2) or 3):

1) the coding sequence is a DNA molecule shown in the 1370-4533 site of the sequence 1;

2) a DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by 1) and codes the fusion protein;

3) a DNA molecule which hybridizes with the nucleotide sequence defined in 1) or 2) under strict conditions and codes for the protein of the fusion protein.

Wherein the DNA molecule may be a cDNA molecule, a genomic DNA molecule or a recombinant DNA molecule.

The DNA molecule of the present invention encoding the above-described fusion protein can be easily mutated by a person of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified to have 75% or more identity to the nucleotide sequence encoding the above-mentioned fusion protein are derived from and identical to the nucleotide sequence of the present invention as long as they encode the above-mentioned fusion protein and have the same function.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence of a protein consisting of the amino acid sequence shown in coding sequence 2 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.

The above stringent conditions are hybridization and washing of the membrane 2 times 5min at 68 ℃ in a solution of 2 XSSC, 0.1% SDS, and hybridization and washing of the membrane 2 times 15min at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS; alternatively, hybridization was carried out at 65 ℃ in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS, and the membrane was washed.

In the method, the encoding gene of the fusion protein is introduced into the receptor cotton through a recombinant vector. The recombinant vector carrying the encoding gene can transform recipient plant cells or tissues by using conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, conductance, Agrobacterium mediation, etc., and culture the transformed plant tissues into plants.

Further, the recombinant vector is a vector obtained by inserting a DNA fragment containing a gene encoding the fusion protein into an expression vector.

Furthermore, the DNA fragment containing the encoding gene of the fusion protein sequentially comprises a promoter for promoting the expression of the encoding gene of the fusion protein, a fluorescent protein mCherry encoding gene and a microtubule positive terminal protein EB1b encoding gene.

In a particular embodiment of the invention, the recombinant vector is specifically pGWB1-P_EB1bmCherry-EB1b, which is the vector obtained by replacing the DNA fragment between attB1 and attB2 sites of pGWB1 expression vector with the DNA molecule shown in sequence 1.

In the above method, the recipient cotton may be a cotton variety commonly found in the prior art, and in a specific embodiment of the present invention, the recipient cotton is new colored cotton No. 7.

The invention also protects the new application of the transgenic cotton or the propagated offspring thereof prepared by the method.

The invention provides application of transgenic cotton or a propagation progeny thereof in any one of the following A1) -A5):

A1) observing the dynamic changes of microtubules in cotton cells;

A2) observing the dynamic changes of microtubules in living cells during the growth and development of cotton;

A3) studying the growth and development of cotton cells;

A4) researching the quality of cotton fiber;

A5) studying the elongation pattern and/or cell division and/or vesicle transport and/or organelle transport and/or cell wall synthesis and/or biotic and abiotic stress response of cotton cells.

In the above application, the cotton cells may be cotton fiber cells; further, the cotton fiber cells can be cotton living fiber cells.

In the application, the propagated progeny comprises the cotton variety, the strain and the mutant with the microtubule positive terminal tag, which are generated by the hybridization of the transgenic cotton obtained by the invention and other cotton varieties, strains, mutants and the like.

The present invention also protects the biomaterial described in any one of 1) to 5) below:

1) the above-mentioned fusion protein;

2) a gene encoding the above fusion protein;

3) an expression cassette, a recombinant vector or a recombinant bacterium containing the encoding gene of the fusion protein;

4) a DNA molecule shown as a sequence 1;

5) a recombinant vector or a recombinant bacterium containing the DNA molecule shown in the sequence 1.

Further, in 4), the recombinant vector is obtained by inserting the DNA molecule shown in sequence 1 into an expression vector;

the recombinant bacterium is a recombinant bacterium containing the recombinant vector.

Further, the recombinant vector may be pGWB1-P_EB1b:mCherry-EB1b；

The recombinant strain contains the pGWB1-P_EB1bAgrobacterium GV3101, mCherry-EB1 b.

The application of the biological material in the preparation of the transgenic cotton also belongs to the protection scope of the invention.

The invention has the beneficial effects that: the invention can be combined with the positive end of a growing micro-tube based on the binding protein EB1b of the positive end of the micro-tube, the positive end of the growing micro-tube in a cotton cell is marked by fluorescent protein, the micro-tube of the cotton cell is marked by specifically tracing the positive end of the growing micro-tube, a transgenic cotton strain is cultivated, and the invention is applied to research on the structure and the dynamic characteristic of the micro-tube in the cotton fiber cell, not only the micro-tube can be marked, but also the dynamic state of the micro-tube can be revealed. Has important significance for analyzing the cotton fiber cell growth mechanism and the cotton fiber genetic improvement.

The invention relates to a recombinant vector pGWB1-P_EB1bmCherry-EB1b is introduced into cotton to obtain transgenic cotton which can express fluorescent protein mCherry labeled microtubulin positive terminal efficiently and stably. The dynamic change of microtubules in living cells in the process of cotton growth and development can be clearly observed by applying a laser confocal imaging system, and the obtained transgenic cotton does not influence the growth and development of cotton, the length of cotton fibers and the yield of cotton, and can be used as a standard strain in the cotton production and research industry. The transgenic cotton obtained by the invention can be used for the research on the aspects of cotton fiber cell elongation mode, cotton fiber growth mechanism, cotton cell growth and development, cell division, vesicle transport, organelle transport, cell wall synthesis, biotic and abiotic stress reaction and the like, and particularly has great application prospect in the aspects of living observation, dynamic analysis and genetic improvement of cotton fiber quality of microtubules in the cotton fiber development process.

Drawings

FIG. 1 is a representation of the phenotype of transgenic cotton and tubulin in leaves and fibers. C7 is wild type new colored cotton No. 7; l1-1-2 is mCherry-EB1b transgenic cotton line. 1a, mCherry-EB1b transgenic cotton line and wild type cotton plant phenotype contrast. 1b, mCherry-EB1b labeled microtubules in cotton fibers. 1c, three-dimensional reconstruction of mCherry-EB1b microtubes in cotton fibers. 1d, the positive end of the microtube in the blade. 1e, microtubes in the blade.

FIG. 2 shows the distribution of the positive ends of microtubules and the arrangement of tubulin in transgenic cotton fibers. 2a, arrangement of microtubules in cotton fiber cells on the day of flowering. 2b, arrangement of microtubules in cotton fiber cells flowering for 2 days. 2c, Kymograph profile of microtubules in cotton fibroblasts on day of flowering. Kymograph plot of microtubules in cotton fibroblasts on two days of flowering. And 2e, a microtubule arrangement model in the cotton fiber development process.

FIG. 3 shows the recombinant plasmid pGWB1-P_EB1bMap of mCherry-EB1 b.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. Modifications or alterations to the methods, steps or conditions of the present invention are within the scope of the invention without departing from the spirit and substance of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

The following examples of Neogossypium hirsutum No. 7 are described in the literature "somatic embryogenesis and plant regeneration in Neogossypium hirsutum No. 7, 2013, Cotton bulletin" and publicly available from the Applicant. The biological material is only used for repeating the related experiments of the invention, and can not be used for other purposes.

pGWB1 expression Vectors in the following examples are described in the "Development OF Series OF gateway Binary Vectors, pGWBs, for the responsive impact Construction OF fusion genes for Plant Transformation, 2007, JOURNAL OF BIOSCIENCE AND DBIOENGINEERING" documents, publicly available from the Applicant. The biological material is only used for repeating the related experiments of the invention, and can not be used for other purposes.

Example 1 acquisition and characterization of transgenic Cotton plants

First, obtaining transgenic cotton plant

1. Construction of recombinant plasmid

pGWB1-P_EB1bThe specific construction method of mCherry-EB1b is as follows: use orderThe DNA molecule shown in column 1 replaced the DNA fragment between the attB1 and attB2 sites of the pGWB1 expression vector to give the recombinant plasmid pGWB1-P_EB1bmCherry-EB1 b. Recombinant plasmid pGWB1-P_EB1bThe map of mCherry-EB1b is shown in figure 3.

The DNA molecule shown in the sequence 1 is formed by connecting a promoter, a fluorescent protein mCherry coding gene and a microtubule positive terminal protein EB1b coding gene in sequence. Wherein, the 1 st-1369 th site of the sequence 1 is a promoter sequence, the 1370 st-2077 th site is a fluorescent protein mCherry coding gene sequence, the 2078 th-2089 th site is a sequence for coding a GAGA joint, and the 2090 st-4533 th site is a microtubule positive terminal protein EB1b coding gene sequence.

Recombinant plasmid pGWB1-P_EB1bmCherry-EB1b expresses fusion protein mCherry-EB1b, the fusion protein mCherry-EB1b is formed by fusing fluorescent protein mCherry and microtubule positive terminal protein EB1b, and the amino acid sequence is shown as sequence 2. The 1 st to 236 th sites of the sequence 2 are the amino acid sequence of the fluorescent protein mCherry, and the 241 nd and 529 th sites are the amino acid sequence of the microtubule positive terminal protein EB1 b.

2. Construction of recombinant bacterium

The recombinant plasmid pGWB1-P prepared in the step 1 is used_EB1bmCherry-EB1b is introduced into Agrobacterium GV3101 (Beijing Bomaide Gene technology Co., Ltd.) to obtain recombinant strain pGWB1-P_EB1b:mCherry-EB1b/GV3101。

3. Transformation of

Adopting an agrobacterium-mediated method to prepare the recombinant bacterium pGWB1-P prepared in the step 2_EB1bmCherry-EB1b/GV3101 is transformed into hypocotyl of No. 7 neocolor cotton, and then cultured and induced in screening culture medium containing hygromycin to form callus, the transformed callus is selected to form embryonic callus in screening culture medium containing hygromycin, and the embryonic callus is cultured to obtain transgenic cotton plant of T0 generation and transgenic cotton plant seed of T1 generation is harvested.

Identification of transgenic cotton plants

1. mCherry-EB1b fluorescence signal detection in true leaves

Planting T1 transgenic cotton plant seeds to obtain T1 transgenic cotton seedlings, observing the first true leaves of the T1 transgenic cotton seedlings by using a rotating disc type confocal microscope, and detecting mCherry-EB1b fluorescent signals (figure 1 d).

2. mCherry-EB1b fluorescence signal detection in other sites

For the plants with fluorescence signals detected in step 1, a rotating disc confocal microscope was used to further detect the mCherry-EB1b fluorescence signals in the cotton fibers (fig. 1b, 1 c). A total of 7 positive transgenic cotton lines were obtained.

3. Additional phenotypic testing of Positive transgenic plants

And observing the growth and development conditions of the positive transgenic cotton plants of the T1 generation. Meanwhile, the wild type new colored cotton No. 7 is used as a control.

The results show that: the phenotype of the obtained positive transgenic line has no significant difference compared with the wild type new colored cotton No. 7, which indicates that the introduction of exogenous DNA molecules does not significantly influence the growth and development of cotton plants.

Example 2 Observation of transgenic Cotton microtubule scaffold

The positive tubulin in plants obtained in example 1 were observed. The method comprises the following specific steps: the method comprises the steps of taking cotton bolls of T1 generation positive transgenic plants 1-3 days after blooming, peeling off peels, taking an ovule, cutting a layer of fiber cells by using an operating blade, placing the fiber cells on a glass slide, adding a cover glass, sealing the fiber cells by using wax, observing mCherry-EB1b signals in the fiber cells by using a rotating disc type confocal microscope, observing the distribution of microtubules marked by EB1b in the cotton fiber cells by using three-dimensional reconstruction at different angles, and continuously shooting EB1b in the cotton fiber cells to reveal the dynamic tissues of the microtubules.

The results show that: the dynamic change of the microtubules in the living cells in the growth and development process of the cotton can be clearly observed by applying a laser confocal imaging system, and the microtubules are arranged in a radial shape in the pores of the cotton leaves (figure 1 e); during the cotton fiber cell protruding period (day of flowering), microtubules are arranged in a scattered manner in cotton fiber cells (fig. 2a, c), and along with the elongation of cotton fibers (two days of flowering), the microtubules are gradually arranged in the fibers to be vertical to the fiber elongation direction (fig. 2b, d), so that a microtubule arrangement model in the cotton fiber development process is obtained (fig. 2 e).

The positive transgenic plant prepared by the invention can be used as a transgenic cotton label strain for observing the dynamic change of the microtubules in the living cotton fiber cells in the cotton growth and development process, and lays a foundation for further analyzing the relation between the cotton fiber growth and development and the microtubule dynamics.

Sequence listing

<110> institute of microbiology of Chinese academy of sciences

<120> cultivation method and application of transgenic cotton tag strain for tracing positive end of microtubule in cotton cell

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Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys His

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Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe

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Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys

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Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys

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Thr Met Gly Trp Glu Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp Gly

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Ala Leu Lys Gly Glu Ile Lys Gln Arg Leu Lys Leu Lys Asp Gly Gly

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His Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly

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Arg His Ser Thr Gly Gly Met Asp Glu Leu Tyr Lys Met Ala Thr Asn

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Ile Gly Met Met Asp Ser Ala Tyr Phe Val Gly Arg Asn Glu Ile Leu

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Ser Trp Ile Asn Asp Arg Leu His Leu Asn Leu Ser Arg Ile Glu Glu

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Ala Ala Ser Gly Ala Val Gln Cys Gln Met Leu Asp Met Thr Phe Pro

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Gly Val Val Pro Met His Lys Val Asn Phe Glu Ala Lys Asn Glu Tyr

290 295 300

Glu Met Ile Gln Asn Tyr Lys Val Met Gln Glu Val Phe Thr Lys Leu

305 310 315 320

Lys Ile Thr Lys Pro Leu Glu Val Asn Arg Leu Val Lys Gly Arg Pro

325 330 335

Leu Asp Asn Leu Glu Phe Leu Gln Trp Leu Lys Arg Phe Cys Asp Ser

340 345 350

Ile Asn Gly Gly Ile Met Asn Glu Asn Tyr Asn Pro Val Glu Arg Arg

355 360 365

Ser Arg Gly Gly Arg Glu Lys Ser Val Lys Gly Ser Ser Lys Ile Ser

370 375 380

Lys Ser Leu Gln Thr Asn Asn Met His His Pro Pro Val Ala Thr Ser

385 390 395 400

Asn Lys Pro Ala Gly Pro Lys Gln Ala Lys Ser His Gly Ile Gly Gly

405 410 415

Gly Ser Asn Ser Ser Ala Glu Val Gln Ala Leu Ser Lys Glu Val Glu

420 425 430

Asp Leu Lys Val Ser Val Asp Leu Leu Glu Lys Glu Arg Asp Phe Tyr

435 440 445

Phe Ser Lys Leu Arg Asp Ile Glu Ile Leu Cys Gln Thr Pro Glu Leu

450 455 460

Asp Asp Leu Pro Ile Val Val Ala Val Lys Lys Ile Leu Tyr Ala Thr

465 470 475 480

Asp Ala Asn Glu Ser Val Leu Glu Glu Ala Gln Glu Cys Leu Asn Gln

485 490 495

Ser Leu Gly Leu Glu Gly Tyr Glu Glu Glu Gly Lys Glu Glu Glu Glu

500 505 510

Glu Glu Glu Glu Glu Glu Glu Glu Ala Ala Ala Ala Ala Glu Thr Gln

515 520 525

Thr

Claims (10)

1. A method for cultivating transgenic cotton comprises the following steps: introducing the encoding gene of the fusion protein into receptor cotton to obtain transgenic cotton;

the fusion protein is formed by fusing fluorescent protein and microtubule positive terminal binding protein EB1 b.

2. The method of claim 1, wherein: the fluorescent protein is a red fluorescent protein mCherry.

3. The method according to claim 1 or 2, characterized in that: the fusion protein is a protein shown in a) or b) or c) or d) as follows:

a) the amino acid sequence is a protein shown in a sequence 2;

b) a fusion protein obtained by connecting a label to the N end and/or the C end of the protein shown in the sequence 2;

c) the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 2;

d) and (b) a protein having a homology of 75% or more than 75% with the amino acid sequence shown in the sequence 2 and having the same function.

4. A method according to any one of claims 1 to 3, wherein: the encoding gene of the fusion protein is the gene shown in the following 1) or 2) or 3):

1) the coding sequence is a DNA molecule shown in the 1370-4533 site of the sequence 1;

2) a DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by 1) and codes the fusion protein;

3) a DNA molecule which hybridizes with the nucleotide sequence defined in 1) or 2) under strict conditions and codes for the protein of the fusion protein.

5. The method according to any one of claims 1 to 4, wherein: the encoding gene of the fusion protein is introduced into receptor cotton through a recombinant vector;

or, the recombinant vector is a vector obtained by inserting a DNA fragment containing the encoding gene of the fusion protein into an expression vector;

or the DNA fragment containing the coding gene of the fusion protein sequentially comprises a promoter for starting the expression of the coding gene of the fusion protein, a fluorescent protein mCherry coding gene and a microtubule positive terminal protein EB1b coding gene.

6. The method of claim 5, wherein: the nucleotide sequence of the DNA fragment of the encoding gene containing the fusion protein is shown as a sequence 1 in a sequence table.

7. The method according to any one of claims 1 to 6, wherein: the acceptor cotton is new colored cotton No. 7.

8. Use of transgenic cotton or its progeny produced by the method according to any one of claims 1 to 7 in any one of the following a1) -a 5):

A1) observing the dynamic changes of microtubules in cotton cells;

A2) observing the dynamic changes of microtubules in living cells during the growth and development of cotton;

A3) studying the growth and development of cotton cells;

A4) researching the quality of cotton fiber;

A5) studying the elongation pattern and/or cell division and/or vesicle transport and/or organelle transport and/or cell wall synthesis and/or biotic and abiotic stress response of cotton cells.

9. The biomaterial according to any one of 1) to 5) below:

1) the fusion protein of claim 3

2) A gene encoding the fusion protein according to claim 4;

3) an expression cassette, a recombinant vector or a recombinant bacterium containing a gene encoding the fusion protein of claim 3;

4) a DNA molecule shown as a sequence 1;

5) a recombinant vector or a recombinant bacterium containing the DNA molecule shown in the sequence 1.

10. Use of the biomaterial of claim 9 in the preparation of the transgenic cotton of any one of claims 1-7.

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