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 label line that traces the positive and extreme ends of microtubules in cotton cells. The method includes the following steps: introducing the encoding gene of the fusion protein into the recipient cotton to obtain the transgenic cotton; the fusion protein is formed by fusing the microtubule positive terminal binding protein EB1b with the fluorescent protein mCherry. The application of laser confocal imaging system can clearly observe the dynamic changes of tubulin in living cells of transgenic cotton during cotton life activities, and the obtained transgenic cotton does not affect the growth and development of cotton, cotton fiber length and cotton yield. Can be used as a standard strain in the cotton production and research industries. The transgenic cotton obtained by the invention can be used for research on cotton growth and development, cell division, organelle transport and the like, especially for the dynamic analysis of microtubules in the cotton fiber development process, and has important application value in the mechanism research of cotton fiber quality improvement.

Description

Culture of a transgenic cotton-tagged line that traces the positive and negative ends of microtubules in cotton cells Breeding methods and their applications

technical field

The invention belongs to the field of biotechnology, and in particular relates to a method for cultivating a transgenic cotton label line that traces the positive and extreme ends of microtubules in cotton cells and its application.

Background technique

Cotton (Gossypium) is one of the most important economic crops in the world and the main source of natural fibers for the global textile industry. The main product of cotton is cotton fiber, and the characteristics 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 the development of cotton fiber cells.

Cotton fiber cells are single cells developed by the specific differentiation and development of epidermal cells of the outer integument of the ovule, and are ideal materials for the study of single cell polar growth and cellulose synthesis. There are two types of cytoskeleton in plant cells: microtubules and microfilaments. Studies have shown that the microtubule skeleton is closely related to the determination of the growth direction of plant cells and cellulose synthesis. Therefore, studying 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 genetic improvement of cotton fibers.

The main component of microtubules is tubulin, and tubulin mainly includes α-tubulin, β-tubulin and microtubule-binding protein. In plant cells, microtubules are in constant change. The subunits at both ends of tubulin are continuously polymerized and depolymerized, and the end with the polymerization speed greater than the depolymerization speed is the positive end of the microtubule.

SUMMARY OF THE INVENTION

The invention uses fluorescent protein to mark the positive end of growing microtubules in cotton cells to obtain transgenic cotton that can observe the microtubule dynamics during the growth of living cotton fiber cells. All leaf epidermal cells, cotton fiber cells and other tissues of the transgenic cotton express mCherry-EB1b fusion protein, and the application of laser confocal imaging system can clearly observe the dynamic changes of microtubules in living cells during cotton growth and development.

The invention firstly protects a method for cultivating a transgenic cotton microtubule label line.

The method for cultivating the transgenic cotton microtubule tag line protected by the present invention comprises the following steps: introducing the encoding gene of the fusion protein into the recipient cotton to obtain the transgenic cotton;

The fusion protein is formed by fusion of microtubule positive end binding protein EB1b and fluorescent protein.

In the above method, the microtubule positive terminal binding protein EB1b (the amino acid sequence is shown in the 241st-529th position of sequence 2) can bind to the positive terminal of the growing microtubule, and express it in cotton cells by fusing it with a fluorescent protein. , the dynamic changes of microtubules in cotton cells can be observed.

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

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

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

b) a fusion protein obtained by linking a tag to the N-terminal and/or C-terminal of the protein shown in SEQ ID NO: 2;

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

d) A protein having 75% or more homology with the amino acid sequence shown in SEQ ID NO: 2 and having the same function.

In order to facilitate purification of the protein in a), a tag as shown in Table 1 can be attached to the amino terminus or carboxyl terminus of the protein shown in SEQ ID NO: 2 in the sequence listing.

Table 1. Sequence of tags

Label Residues sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

For the protein in the above c), the substitution and/or deletion and/or addition of one or several amino acid residues are substitutions and/or deletions and/or additions of no more than 10 amino acid residues.

The protein in the above c) can be obtained by artificial synthesis, or by first synthesizing its encoding gene and then biologically expressing it.

The coding gene of the protein in the above c) can be obtained by deleting the codon of one or several amino acid residues in the DNA sequence shown in SEQ ID NO: 1, and/or carrying out missense mutation of one or several base pairs, and/ Or the coding sequence of the tag shown in Table 1 is attached to its 5' end and/or 3' end.

In the above d), "homology" includes 75% or more, or 80% or more, or 85% or more, or 90% or more with the amino acid sequence shown in SEQ ID NO: 2 of the present invention, or amino acid sequences with 95% or higher homology.

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

1) Its coding sequence is the DNA molecule shown in the 1370th-4533rd position of sequence 1;

2) a DNA molecule that has 75% or more identity with the nucleotide sequence defined in 1) and encodes the fusion protein;

3) A DNA molecule that hybridizes under stringent conditions to the nucleotide sequences defined in 1) or 2) and encodes the protein of the fusion protein.

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

Those of ordinary skill in the art can easily use known methods, such as directed evolution and point mutation methods, to mutate the DNA molecules encoding the above fusion proteins of the present invention. Those artificially modified nucleotides with 75% or higher identity to the nucleotide sequence encoding the above-mentioned fusion protein, as long as they encode the above-mentioned fusion protein and have the same function, are all derived from the nucleotide sequence of the present invention and Equivalent to the sequences of the present invention.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "Identity" includes 75% or higher, or 85% or higher, or 90% or higher, or 95% or Nucleotide sequences of higher identity. Identity can be assessed with the naked eye or with 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 75% or more identity may be 80%, 85%, 90% or more than 95% identity.

The above stringent conditions were hybridized in a solution of 2 × SSC, 0.1% SDS at 68 °C and washed twice for 5 min, and then in a solution of 0.5 × SSC, 0.1% SDS at 68 °C. Wash the membrane twice for 15 min each; or, hybridize and wash the membrane in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS at 65°C.

In the above method, the gene encoding the fusion protein is introduced into the recipient 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, electrical conductivity, and Agrobacterium-mediated transformation, and The transformed plant tissue is grown into plants.

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

Further, the DNA fragment containing the gene encoding the fusion protein sequentially includes a promoter for initiating the expression of the gene encoding the fusion protein, a gene encoding a fluorescent protein mCherry, and a gene encoding a microtubule positive terminal protein EB1b.

In a specific embodiment of the present invention, the recombinant vector is specifically pGWB1-P _EB1b :mCherry-EB1b, which is to replace the DNA fragment between the attB1 and attB2 sites of the pGWB1 expression vector with the DNA molecule shown in sequence 1. the resulting vector.

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

Another aspect of the present invention protects the new use of the transgenic cotton prepared according to the above method or its reproductive progeny.

The invention provides the application of transgenic cotton or its breeding progeny in any one of the following A1)-A5):

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

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

A3) study the growth and development of cotton cells;

A4) study the quality of cotton fibers;

A5) Study 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 responses of cotton cells.

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

In the above application, the breeding offspring include cotton varieties, lines and mutants with microtubule positive and extreme labels produced by crossing the transgenic cotton obtained in the present invention with other cotton varieties, lines, mutants, etc.

The present invention also protects the biological material described in any of the following 1)-5):

1) the above fusion protein;

2) the encoding gene of the above-mentioned fusion protein;

3) an expression cassette, a recombinant vector or a recombinant bacteria containing the encoding gene of the above-mentioned fusion protein;

4) the DNA molecule shown in sequence 1;

5) A recombinant vector or recombinant bacteria containing the DNA molecule shown in SEQ ID NO: 1.

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

The recombinant bacteria are recombinant bacteria containing the recombinant vector.

Further, the recombinant vector can be pGWB1-P _EB1b :mCherry-EB1b;

The recombinant bacteria is Agrobacterium GV3101 containing the pGWB1-P _EB1b :mCherry-EB1b.

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

Beneficial effects of the present invention: The present invention is based on the fact that the microtubule positive terminal binding protein EB1b can bind to the positive terminal of the growing microtubule, and uses fluorescent protein to mark the positive terminal of the growing microtubule in cotton cells, and specifically traces the growing microtubule. The positive end of microtubules can be used to label cotton cell microtubules, cultivate transgenic cotton lines, and be applied to study the structure and dynamic characteristics of microtubules in cotton fiber cells. It can not only label microtubules, but also reveal the dynamics of microtubules. It is of great significance for analyzing the growth mechanism of cotton fiber cells and the genetic improvement of cotton fiber.

In the present invention, the recombinant vector pGWB1-P _EB1b :mCherry-EB1b is introduced into cotton to obtain a transgenic cotton that efficiently and stably expresses the positive end of the tubulin labeled with the fluorescent protein mCherry. The application of laser confocal imaging system can clearly observe the dynamic changes of microtubules in living cells during the growth and development of cotton, and the obtained transgenic cotton does not affect the growth and development of cotton, cotton fiber length and cotton yield, and can be used as cotton production and cotton production. Research industry standard strains. The transgenic cotton obtained by the present invention can be used for the research on the cell elongation mode of cotton fiber, the growth mechanism of cotton fiber, cotton cell growth and development, cell division, vesicle transport, organelle transport, cell wall synthesis, biotic and abiotic stress responses, etc. , especially in the in vivo observation and dynamic analysis of microtubules during cotton fiber development and the genetic improvement of cotton fiber quality.

Description of drawings

Figure 1. Transgenic cotton phenotype and tubulin in leaves and fibers. C7 is the wild type Xincaimian No. 7; L1-1-2 is the mCherry-EB1b transgenic cotton line. 1a. Phenotypic comparison of mCherry-EB1b transgenic cotton lines and wild-type cotton plants. 1b, mCherry-EB1b-labeled microtubules in cotton fibers. 1c, 3D reconstruction of microtubules of mCherry-EB1b in cotton fibers. 1d. Positive end of microtubules in leaves. 1e, Microtubules in leaves.

Figure 2 shows the distribution of the positive end of microtubules and the arrangement of tubulin in transgenic cotton fibers. 2a. The arrangement of microtubules in cotton fiber cells on the day of flowering. 2b, Arrangement of microtubules in cotton fiber cells for 2 days after flowering. 2c, Kymograph of microtubules in cotton fiber cells on the day of flowering. 2d, Kymograph map of microtubules in cotton fiber cells two days after flowering. 2e, Microtubule arrangement model during cotton fiber development.

Figure 3 is a map of the recombinant plasmid pGWB1-P _EB1b :mCherry-EB1b.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. Modifications or substitutions of the methods, steps or conditions of the present invention without departing from the spirit and essence of the present invention all belong to the scope of the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples are all set to repeat the experiments three times, and the results are averaged.

The new color cotton No. 7 in the following examples is described in the document "Somatic embryogenesis of green cotton new color cotton No. 7 and its plant regeneration, 2013, Cotton Journal", and the public can obtain it from the applicant. The biological material is only used for repeating the relevant experiments of the present invention, and cannot be used for other purposes.

The pGWB1 expression vector in the following examples is described in the document "Development of Series of Gateway Binary Vectors, pGWBs, for Realizing Efficient Construction of FusionGenes for Plant Transformation, 2007, JOURNAL OF BIOSCIENCE ANDBIOENGINEERING", which is publicly available from the applicant. The biological material is only used for repeating the relevant experiments of the present invention, and cannot be used for other purposes.

Example 1. Acquisition and identification of transgenic cotton plants

First, the acquisition of transgenic cotton plants

1. Construction of recombinant plasmids

The specific construction method of pGWB1-P _EB1b :mCherry-EB1b is as follows: replace the DNA fragment between the attB1 and attB2 sites of the pGWB1 expression vector with the DNA molecule shown in sequence 1 to obtain the recombinant plasmid pGWB1-P _EB1b :mCherry-EB1b. The map of the recombinant plasmid pGWB1-P _EB1b :mCherry-EB1b is shown in Figure 3.

The DNA molecule shown in sequence 1 is formed by connecting the promoter, the fluorescent protein mCherry encoding gene and the microtubule positive terminal protein EB1b encoding gene in turn. The 1-1369th position of sequence 1 is the promoter sequence, the 1370-2077th position is the fluorescent protein mCherry coding gene sequence, the 2078th-2089th position is the sequence encoding the GAGA linker, and the 2090th-4533th position is the microtubule positive extremity protein EB1b coding gene sequence.

The recombinant plasmid pGWB1-P _EB1b : mCherry-EB1b expresses the fusion protein mCherry-EB1b. The fusion protein mCherry-EB1b is a protein formed by the fusion of the fluorescent protein mCherry and the microtubule positive and extreme protein EB1b, and its amino acid sequence is shown in sequence 2. Positions 1-236 of sequence 2 are the amino acid sequence of fluorescent protein mCherry, and positions 241-529 are the amino acid sequence of microtubule positive extremity protein EB1b.

2. Construction of recombinant bacteria

The recombinant plasmid pGWB1-P _EB1b : mCherry-EB1b prepared in step 1 was introduced into Agrobacterium GV3101 (Beijing Biomed Gene Technology Co., Ltd.) to obtain the recombinant bacterium pGWB1-P _EB1b : mCherry-EB1b/GV3101.

3. Conversion

The recombinant strain pGWB1-P _EB1b :mCherry-EB1b/GV3101 prepared in step 2 was transformed into the hypocotyl of Xincai Mian No. 7 by the method mediated by Agrobacterium, and then cultured in the selection medium containing hygromycin to induce Callus is formed, transformed callus is selected, embryogenic callus is formed from the callus in the selection medium containing hygromycin, cultured to obtain T0 generation transgenic cotton plants, and T1 generation transgenic cotton plant seeds are harvested.

2. Identification of transgenic cotton plants

1. Detection of mCherry-EB1b fluorescence signal in true leaves

The seeds of T1 generation transgenic cotton plants were planted to obtain T1 generation transgenic cotton seedlings. The first true leaf of the T1 generation transgenic cotton seedlings was observed with a rotating disk confocal microscope, and the mCherry-EB1b fluorescence signal was detected (Fig. 1d).

2. Detection of mCherry-EB1b fluorescence signal in other parts

For plants whose fluorescence signal was detected in step 1, the mCherry-EB1b fluorescence signal in cotton fiber was further detected by a spinning disk confocal microscope (Figure 1b, 1c). A total of 7 positive transgenic cotton lines were obtained.

3. Other phenotypic detection of positive transgenic plants

The growth and development of T1 generation positive transgenic cotton plants were observed. At the same time, wild-type Xincaimian No. 7 was used as the control.

The results showed that the phenotype of the obtained positive transgenic line was not significantly different from that of the wild type Xincaimian 7, indicating that the introduction of exogenous DNA molecules did not significantly affect the growth and development of cotton plants.

Example 2. Observation of transgenic cotton microtubule skeleton

The microtubule positive and extreme proteins in the positive transgenic plants obtained in Example 1 were observed. The specific steps are as follows: take the cotton bolls of the T1 generation positive transgenic plants 1-3 days after flowering, peel off the pericarp, take an ovule, cut a layer of fiber cells with a surgical blade, place it on a glass slide, add a cover glass, and use After wax sealing, the mCherry-EB1b signal in fibroblasts was observed by a spinning disk confocal microscope, the distribution of EB1b-labeled microtubules in cotton fibroblasts was observed by 3D reconstruction from different angles, and the EB1b in cotton fibroblasts was continuously photographed to reveal Dynamic organization of microtubules.

The results showed that the application of laser confocal imaging system can clearly observe the dynamic changes of microtubules in living cells during cotton growth and development. In cotton leaf stomata, microtubules are arranged radially (Fig. 1e); (on the day of flowering), microtubules were scattered in cotton fiber cells (Fig. 2a, c), and with the elongation of cotton fibers (two days after flowering), the microtubules in the fibers were gradually arranged perpendicular to the direction of fiber elongation (Fig. 2a, c). 2b, d), thereby obtaining a model of microtubule arrangement during cotton fiber development (Fig. 2e).

The positive transgenic plants prepared by the invention can be used as transgenic cotton label lines to observe the dynamic changes of microtubules in living cotton fiber cells in the process of cotton growth and development, and lay a foundation for further analysis of the relationship between cotton fiber growth and development and microtubule dynamics .

sequence listing

<110> Institute of Microbiology, Chinese Academy of Sciences

<120> A method for cultivating a transgenic cotton label line that traces the positive and negative ends of microtubules in cotton cells and its application

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<170>PatentIn version 3.5

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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, comprising the steps of: introducing the encoding gene of a fusion protein into recipient cotton to obtain the transgenic cotton; The fusion protein is formed by fusion of fluorescent protein and microtubule positive terminal binding protein EB1b. 2. The method according to claim 1, wherein the fluorescent protein is red fluorescent protein mCherry. 3. The method according to claim 1 or 2, wherein the fusion protein is the protein shown in the following a) or b) or c) or d): a) the amino acid sequence is the protein shown in sequence 2; b) a fusion protein obtained by linking a tag to the N-terminal and/or C-terminal of the protein shown in SEQ ID NO: 2; c) a protein with the same function obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in Sequence 2; d) A protein having 75% or more homology with the amino acid sequence shown in SEQ ID NO: 2 and having the same function. 4. according to the arbitrary described method of claim 1-3, it is characterized in that: the coding gene of described fusion protein is the gene shown in following 1) or 2) or 3): 1) Its coding sequence is the DNA molecule shown in the 1370th-4533th position of sequence 1; 2) a DNA molecule that has 75% or more identity with the nucleotide sequence defined in 1) and encodes the fusion protein; 3) A DNA molecule that hybridizes under stringent conditions to the nucleotide sequences defined in 1) or 2) and encodes the protein of the fusion protein. 5. The method according to any one of claims 1-4, wherein the encoding gene of the fusion protein is introduced into the recipient cotton through a recombinant vector; Or, the recombinant vector is a vector obtained by inserting a DNA fragment containing a gene encoding a fusion protein into an expression vector; Or, the DNA fragment containing the gene encoding the fusion protein sequentially includes a promoter for initiating the expression of the gene encoding the fusion protein, a gene encoding a fluorescent protein mCherry, and a gene encoding a microtubule positive and extreme protein EB1b. 6 . The method according to claim 5 , wherein the nucleotide sequence of the DNA fragment of the encoding gene containing the fusion protein is shown in SEQ ID NO: 1 in the sequence listing. 7 . 7. The method according to any one of claims 1-6, wherein the acceptor cotton is No. 7 new colored cotton. 8. the application of the transgenic cotton prepared according to the arbitrary described method of claim 1-7 or its breeding offspring in any one of following A1)-A5): A1) observe the dynamic changes of microtubules in cotton cells; A2) observe the dynamic changes of microtubules in living cells during cotton growth and development; A3) study the growth and development of cotton cells; A4) study the quality of cotton fibers; A5) Study the elongation patterns and/or cell division and/or vesicle trafficking and/or organelle trafficking and/or cell wall synthesis and/or biotic and abiotic stress responses of cotton cells. 9. The biological material described in any of the following 1)-5): 1) fusion protein described in claim 3 2) the coding gene of the fusion protein described in claim 4; 3) an expression cassette, a recombinant vector or a recombinant bacteria containing the encoding gene of the fusion protein described in claim 3; 4) the DNA molecule shown in sequence 1; 5) A recombinant vector or recombinant bacteria containing the DNA molecule shown in SEQ ID NO: 1. 10. Use of the biological material according to claim 9 in the preparation of the transgenic cotton according to any one of claims 1-7.

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