阅读说明：本技术 一种标记棉花细胞微丝骨架的转基因棉花标签株系的培育方法及其应用 (Cultivation method and application of transgenic cotton tag strain for marking cotton cell microfilament skeleton ) 是由 孔照胜 于艳军 吴慎杰 王光达 田娟 马银平 于 2019-02-21 设计创作，主要内容包括：本发明公开了一种标记棉花细胞微丝骨架的转基因棉花标签株系的培育方法及其应用。所述培育方法包括如下步骤：将融合蛋白的编码基因导入受体棉花中,得到所述转基因棉花；所述融合蛋白由拟南芥微丝结合蛋白Fimbrin-1的第二个微丝结合结构域ABD2与绿色荧光蛋白GFP融合而成。应用激光共聚焦成像系统能清晰地观察在棉花生长发育过程中活细胞中的微丝的动态变化,可用作为棉花生产和研究行业的标准株系。本发明获得的转基因棉花标签株系可用于棉花纤维生长机理、生长发育、细胞分裂、囊泡运输、细胞器运输等方面的研究,尤其对于棉花纤维发育过程中微丝的观察、分析和棉花纤维品质的提高都有重大的应用前景。(The invention discloses a cultivation method and application of a transgenic cotton tag strain for marking a cotton cell microfilament skeleton. The cultivation 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 second microfilament binding structural domain ABD2 of an arabidopsis microfilament binding protein Fimbrin-1 and a green fluorescent protein GFP. The dynamic change of microfilaments in living cells in the growth and development process of cotton can be clearly observed by using a laser confocal imaging system, and the method can be used as a standard strain of cotton production and research industries. The transgenic cotton label strain obtained by the invention can be used for the research on the aspects of cotton fiber growth mechanism, growth and development, cell division, vesicle transportation, organelle transportation and the like, and particularly has great application prospect on the observation and analysis of microfilaments in the cotton fiber development process and the improvement of the quality of cotton fibers.)

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 a second microfilament binding structural domain ABD2 of an arabidopsis microfilament binding protein Fimbrin-1 and a fluorescent protein.

2. The method of claim 1, wherein: the fluorescent protein is green fluorescent protein GFP.

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 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 acceptor cotton through a recombinant vector.

6. The method of claim 5, wherein: the recombinant vector is pCAMBIA1390-ABD 2-GFP.

7. The method according to any one of claims 1 to 6, wherein: the acceptor cotton is upland cotton R15.

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 change of microfilaments in cotton cells;

A2) observing the dynamic change of microfilaments 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 3) below:

1) a fusion protein as set forth in claim 3;

2) the fusion protein of claim 4, wherein the fusion protein encodes a gene

3) An expression cassette, a recombinant vector or a recombinant bacterium containing a gene encoding the fusion protein of claim 4.

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

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 marking a cotton cell microfilament framework.

Background

Cotton (Gossypium) is one of the world's important commercial crops, and is second only to grains. Cotton is composed mainly of cotton fibers, and the properties of cotton fibers 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. Studies have shown that the cytoskeleton plays an extremely important role in fibroblast elongation and cellulose synthesis.

The main component of the microfilament is actin, which is polymerized with each other in the form of subunits to form a helical structure, and the structure is in a constantly changing state. Research shows that microfilaments play a regulating role in the processes of cell division, differentiation and development, and the expression change of related genes influences the fiber cell elongation. However, the previous researches show that all the microwires in fiber cells are fixed based on an observed chemical method, if the dynamic change of the microwires in living cotton fiber cells in the fiber development process can be observed, the deep understanding of the cotton fiber cell development mechanism can be greatly promoted, and meanwhile, the method has important significance for improving the quality of cotton fibers.

Disclosure of Invention

The invention utilizes green fluorescent protein GFP to mark the second microfilament binding domain ABD2 of arabidopsis thaliana microfilament binding protein Fimbrin-1 to transform cotton, and obtains transgenic cotton capable of observing the dynamic change of microfilaments in the growth process of cotton living fiber cells. All leaf epidermal cells, sepal cells, filament cells, root cells and cotton fiber cells of the transgenic cotton plant are expressed by ABD2-GFP fusion protein, and the dynamic change of microfilaments 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 cultivation method of transgenic cotton.

The method for cultivating the 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 a second microfilament binding structural domain ABD2 of an arabidopsis microfilament binding protein Fimbrin-1 and a fluorescent protein.

In the method, the amino acid sequence of the second microfilament binding domain ABD2 of the Arabidopsis thaliana microfilament binding protein Fimbrin-1 is shown as 1 st-339 th sites of a sequence 2.

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 green fluorescent protein GFP, and the fusion protein is formed by fusing the second microwire binding domain ABD2 of the arabidopsis microwire binding protein Fimbrin-1 and the green fluorescent protein GFP.

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 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 pCAMBIA1390-ABD2-GFP, which is described in "organization of microorganisms and the action cytokine in a plasmid cell determination by a plant-unique kinase, 2015, Elife".

In the method, the recipient cotton can be a cotton variety which is common in the prior art. In one embodiment of the invention, the recipient cotton is upland cotton R15.

The recombinant vector pCAMBIA1390-ABD2-GFP is transferred into an agrobacterium EHA105 strain, and the agrobacterium transformation method is utilized to obtain the transgenic cotton marked with the cotton microfilament binding protein.

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 change of microfilaments in cotton cells;

A2) observing the dynamic change of microfilaments 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 cotton varieties, lines and mutants with actin microfilament labels, which are generated by the hybridization of the transgenic cotton obtained by the invention and other cotton varieties, lines, mutants and the like.

The present invention also protects the biomaterial described in any one of 1) to 3) 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.

Further, in the 3), the recombinant vector can be pCAMBIA1390-ABD 2-GFP;

the recombinant strain can be agrobacterium EHA105 containing the pCAMBIA1390-ABD 2-GFP.

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

The invention introduces pCAMBIA1390-ABD2-GFP into cotton to obtain transgenic cotton which efficiently and stably expresses fluorescent protein GFP labeled actin microfilament binding protein. The dynamic change of microfilaments in living cells in the cotton growth and development process can be clearly observed by using a laser confocal imaging system, the growth and development of the obtained transgenic cotton, the cotton fiber length, the cotton yield and the like are not influenced, and the transgenic cotton 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 on the observation and analysis of microfilaments in the cotton fiber development process and the improvement of cotton fiber quality.

Drawings

FIG. 1 shows actin microfilaments in transgenic cotton phenotypes and different organs. a. Compared with the phenotype of a wild cotton plant, the ABD2-GFP transgenic cotton line has the characteristics that R-15 is wild upland cotton R15, and L8-83 and L9-16 are ABD2-GFP transgenic cotton lines. b. Comparison of fiber length of ABD2-GFP transgenic Cotton with wild-type Cotton. c. ABD2-GFP transgenic Cotton root, leaf, epidermal hair, filament, corolla, and microfilament morphology and distribution in cotton fiber.

FIG. 2 shows the arrangement of microfilaments in transgenic cotton fiber cells. And (3) arranging microfilaments in the ABD2-GFP transgenic cotton fiber at the left side 1, and reconstructing a different angle map in the microfilaments in the ABD2-GFP transgenic cotton fiber at the left side 2-4.

FIG. 3 shows actin microfilament dynamics in transgenic cotton fiber cells. And the morphological structure of the microfilament in the ABD2-GFP transgenic cotton fiber at the left 1, and the dynamic time sequence development chart of the microfilament in the ABD2-GFP transgenic cotton fiber at the left 2-5.

FIG. 4 shows the morphology and distribution of actin microfilaments in transgenic cotton leaves.

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 cotton Plant R15 in the following examples is described in "signalling Improvement of cottonVerticillium wild Resistance by Manipulating the Expression of chemical Proteins, 2016, Molecular Plant", 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.

The recombinant vector pCAMBIA1390-ABD2-GFP described in the following examples is described in the literature "engineering of microorganisms and the enzyme cytoskeleton in a chromatograph cell shape determination by a plant-unique kinase, 2015, Elife", and is 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.