Application of cotton GhMAF1 gene in fiber yield regulation and control

Abstract

The invention provides application of a cotton GhMAF1 gene in fiber yield regulation and control, and the gene for regulating and controlling cotton fiber development is the GhMAF1 gene and comprises 672 nucleotide sequences. According to the invention, a GhMAF1 gene knockout vector is constructed and cotton is transformed, the analysis on the GhMAF1 gene knockout cotton shows that the number of fibers of the GhMAF1 gene knockout cotton is obviously reduced, and the GhMAF1 gene can be applied to the improvement of the development process of cotton fibers, so that a special function is exerted or a special transgenic plant material is created.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically, to the application of cotton GhMAF1 gene in regulating fiber yield. Background Art

[0002] Cotton (Gossypium spp.) is one of the world's major economic crops and the main source of natural fibers for the textile industry. Cotton fibers are the fibers covered on its seeds and are developed from the epidermal cells of fertilized ovules. The cotton fiber development process is divided into four stages: initiation, elongation, secondary cell wall thickening, and fiber maturation.

[0003] Genes support the basic structure and properties of life. They store all the information of the processes such as the race, blood type, gestation, growth, apoptosis, etc. of life. The mutual dependence of environment and heredity interprets important physiological processes such as the reproduction of life, cell division, and protein synthesis. All life phenomena such as the birth, growth, decline, disease, aging, and death of organisms are related to genes.

[0004] Cotton fibers are the main source of natural textile fibers worldwide. With the improvement of living standards, people's requirements for high-grade fiber textiles are also getting higher and higher. Therefore, improving fiber yield and quality is one of the main goals of current cotton breeding. Due to the long cycle and many limitations of traditional breeding methods, and the difficulty in stabilizing the strains obtained by hybridization, it is of great significance to use genetic engineering methods to directionally improve fiber quality. Summary of the Invention

[0005] The present invention provides the gene GhMAF1 that regulates cotton fiber development, which includes a nucleotide sequence of 672. The present invention constructs a GhMAF1 gene knockout vector and transforms cotton. Through the analysis of GhMAF1 gene knockout cotton, it is found that the number of its fibers is significantly reduced, and it can be applied to improve the cotton fiber development process, thereby playing a special function or creating special transgenic plant materials.

[0006] The technical solution of the present invention is as follows:

[0007] The first object of the present invention is to provide an application of GhMAF1 protein, its coding gene, or a recombinant vector, expression cassette, transgenic cell line, or recombinant bacterium containing a DNA molecule encoding GhMAF1 protein in regulating cotton fiber yield.

[0008] Preferably, in any of the above-mentioned solutions, the coding gene of GhMAF1 protein is as shown in Sequence 1.

[0009] Preferably, in any of the above-mentioned solutions, the amino acid sequence of GhMAF1 protein is as shown in Sequence 2.

[0010] Preferably, in any of the above solutions, the sequence 1 regulates cotton fiber and promotes an increase in fiber quantity.

[0011] Preferably, in any of the above solutions, the sequence for amplifying the GhMAF1 coding gene includes an upstream sequence and a downstream sequence. The upstream sequence is as shown in sequence 3, and the downstream sequence is as shown in sequence 4.

[0012] The second object of the present invention is to provide a method for promoting an increase in cotton fiber yield, including the following steps: introducing the coding gene of the GhMAF1 protein into a target plant, increasing the expression or activity of the GhMAF1 protein in the target plant to obtain a transgenic plant, where the target plant is cotton.

[0013] Preferably, the coding gene of the GhMAF1 protein is as shown in sequence 1.

[0014] Preferably, the amino acid sequence of the GhMAF1 protein is as shown in sequence 2.

[0015] The present invention constructed a GhMAF1 gene knockout vector and transformed cotton. Through the analysis of the GhMAF1 gene knockout cotton, compared with the wild-type cotton, it was found that the number of fibers in the knockout cotton was significantly reduced. This shows that the GhMAF1 gene regulates the development of cotton fibers, providing a theoretical basis for cotton variety improvement and can be used to cultivate new cotton varieties with excellent fiber yield and quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the embodiments. It should be understood that the following drawings only show the embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0017] Figure 1 It is a graph showing the expression results of GhMAF1 in different tissues of cotton detected by qRT-PCR;

[0018] Figure 2 It is the vector used for obtaining the GhMAF1 cotton mutant material;

[0019] Figure 3 It is a schematic diagram showing that sgRNA1 and sgRNA2 are respectively located in the first and second exons of GhMAF1;

[0020] Figure 4 It is a graph showing the sequencing analysis results in the obtaining of the GhMAF1 cotton mutant material;

[0021] Figure 5Results of the expression levels of GhMAF1 in mutants m1 and m2 and ZM24;

[0022] Figure 6 Development of fibroblast cells on the basal epidermal cells of 0, 2, and 5 DPA ovules of mutants m1 and m2 and ZM24;

[0023] Figure 7 Fiber development at the base of seeds of mutants m1 and m2 and ZM24;

[0024] Figure 8 Results of the total fiber amount on the seed surface of mutants m1 and m2 and ZM24; Detailed implementation method

[0025] Unless otherwise specified, the methods used in the following examples are all conventional methods.

[0026] Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained from commercial channels.

[0027] Example

[0028] Cloning and sequencing of the GhMAF1 gene

[0029] 1. Extract total DNA from Gossypium hirsutum L. cv. Zhongmian 24 (ZM24) plants. Using this as a template,

[0030] Primer GhMAF1-F 5’-ATGAAGTTCCTAGAATACAC-3’

[0031] Primer GhMAF1-R 5’-TTACATGTCCATGTTATCAA-3’

[0032] 2. Perform PCR amplification and PCR product sequencing on the GhMAF1 gene (Ghicr24_A07G192800). Its gene coding sequence and amino acid sequence are as follows:

[0033] Gene coding sequence:

[0034] ATGAAGTTCCTAGAATACACTCCATTTGACCGAATAAATGATTTCTTGAGAGAACTAAACCTTGGTGAACGTACCATCAAAGGGAGCCTTGAAGCATACTCTTGCAAACACACAGGAACTGATAAAAGACTTTCTCTCAGTTTGGAAAATGAGATCCTTGACTCTCTTGGGAAATCTTCAGATACCGACTCCTCACCAGTTGAATTCCTGTTGAGCAGATCAAGCCGGAAGACGTTGATCTACTTGGTGCTTACCCTCTATCACATGTATCCAGATTATGACTTAAGTGCAGTTAAAGCACACCAGTTCTTCACTGAGGAAATCTGGGATACTTTTAAGCAGATTTTTGAAACCTACATGCTTGAAGCCTCAAAGGAATGGATTGAGACATACGGGGGCAGTTCACTCTTGGAAACTGTGTATAAGGCTCTAGATGAGGTTGTGAATCTCTCGGAGTGTGAAATTTACAGTTATAATCCGGACTCTGATGCAGATCCCTTTCTGGAGAAAGGGGCAATATGGTCCTTCAGTTTCTTCTTTTACAATAGAAAGTTGAAACGTGTTGTGAGTTTCAGCTTTTGCTGCTTAAGTAATTTGGTGGGTGATGGATTCCTTGCTGACAACTTATGTTCTGAGGAAGATGGTGAAATATTTGATAACATGGACATGTAAA。

[0035] Amino acid sequence:

[0036] MKFLEYTPFDRINDFLRELNLGERTIKGSLEAYSCKHTGTDKRLSLSLENEILDSLGKSSDTDSSPVEFLLSRSSRKTLIYLVLTLYHMYPDYDLSAVKAHQFFTEEIWDTFKQIFETYMLEASKEWIETYGGSSLLETVYKALDEVVNLSECEIYSYNPDSDADPFLEKGAIWSFSFFFYNRKLKRVVSFSFCCLSNLVGDGFLADNLCSEEDGEIFDNMDM。

[0037] Tissue expression pattern of GhMAF1 in cotton

[0038] The tissue expression pattern of a gene reflects the function of the gene to a certain extent. The expression of GhMAF1 in different tissues of cotton was detected by qRT-PCR (as Figure 1 shown). The results showed that at the fiber initiation stage (-3, 0, 1 DPA), the expression level of GhMAF1 was higher in ovules, and fiber cells are differentiated from the ovule epidermal cells during this period, indicating that GhMAF1 plays a role in fiber development.

[0039] Obtaining GhMAF1 cotton mutant materials

[0040] To verify the biological function of GhMAF1, the CRISPR / Cas9 gene editing technology was used to create GhMAF1 cotton mutant materials m1 and m2. The vector used was pRGEB32-GhU6.7-NPT II (as Figure 2 shown), and sgRNA1 and sgRNA2 were located on the first and second exons of GhMAF1 respectively (as Figure 3 shown). Sequencing analysis of GhMAF1 showed that a single-base deletion in sgRNA1 and a three-base deletion in sgRNA2 resulted in premature termination of translation of GhMAF1 after only 12 amino acids in m1. Inserting a single base at sgRNA1 and sgRNA2 respectively led to premature termination of translation of GhMAF1 after only 9 amino acids in m2 (as Figure 4 shown). Through qRT-PCR analysis, it was found that the expression level of GhMAF1 in m1 and m2 was significantly lower than that in ZM24 (as Figure 5 shown).

[0041] Phenotypes of GhMAF1 cotton mutant materials during the fiber initiation and development period

[0042] After fiber initiation analysis, fiber cells were only observed at the top of the ovules, but not on the basal epidermal cells of the 0, 2, and 5 DPA ovules of m1 and m2, while fiber cells usually initiate and elongate on the entire ovule surface of ZM24 (as Figure 6 shown).

[0043] Phenotypes of GhMAF1 cotton mutant materials during the fiber maturation period

[0044] There was a lack of fiber development at the base of m1 and m2 seeds, and there was also a lack of lint cells (as Figure 7 shown). Compared with the wild type ZM24, the total fiber amount on the surface of each seed of m1 and m2 mutants was significantly reduced (as Figure 8 shown), indicating that GhMAF1 regulates the development and yield of cotton fibers.

[0045] In summary, the GhMAF1 gene regulates the development of cotton fibers, can effectively increase the number of cotton fibers, provides a theoretical basis for the variety improvement of cotton, and can be used to cultivate new cotton varieties with excellent fiber yield and quality.

[0046] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of GhMAF1 protein, its coding gene, recombinant vector containing DNA molecule encoding GhMAF1 protein, expression cassette, transgenic cell line or recombinant bacterium in regulating cotton fiber yield.

2. The application according to claim 1, wherein The coding gene of GhMAF1 protein is as shown in Sequence 1.

3. The application according to claim 1, characterized in that, The amino acid sequence of GhMAF1 protein is as shown in Sequence 2.

4. The application according to claim 1, characterized in that The said Sequence 1 regulates the number of cotton fibers and promotes the increase in fiber number.

5. The application according to claim 1, characterized in that, The sequence for amplifying the coding gene of GhMAF1 includes an upstream sequence and a downstream sequence. The upstream sequence is as shown in Sequence 3, and the downstream sequence is as shown in Sequence 4.

6. A method for promoting the increase in cotton fiber yield, comprising the following steps: introducing the coding gene of GhMAF1 protein into a target plant, enhancing the expression or activity of GhMAF1 protein in the target plant to obtain a transgenic plant, and the target plant is cotton.

7. The method according to claim 6, wherein The coding gene of the said GhMAF1 protein is as shown in Sequence 1.

8. The method according to claim 6, wherein The amino acid sequence of the said GhMAF1 protein is as shown in Sequence 2.

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