Application of cotton GhSAC7 protein in regulation and control of initial differentiation of cotton fibers

Abstract

The invention discloses a cotton GhSAC7 protein coding gene (GhD03G0020, LOC107937826), the coding sequence of the cotton GhSAC7 protein coding gene is 1791 nucleotides, and the protein sequence of the cotton GhSAC7 protein coding gene is 596 amino acids. Protein expression level analysis and phenotype analysis are carried out on a gene editing material of the GhSAC7 gene, and results show that the gene editing material of the GhSAC7 gene has phenotypes of slowing down fiber initiation, reducing fiber density and reducing total fiber amount. Therefore, the GhSAC7 can effectively regulate the development of the cotton fibers and promote the start of the cotton fibers, provides a theoretical basis for the variety improvement of the cotton, and can be used for cultivating new cotton varieties with excellent fiber quality.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, and in particular to application of cotton GhSAC7 protein in regulating the initial differentiation of cotton fibers. Background Art

[0002] Cotton (Gossypium spp.) is one of the world's most important economic crops, and its seed coat fibers (cotton fibers) are a crucial raw material for the textile industry. Cotton fiber development is divided into four stages: initiation, elongation, secondary cell wall deposition, and fiber maturation. The initiation and elongation stages determine the number and length of cotton fibers. Therefore, studying the molecular mechanisms of these stages is of great practical significance for improving cotton yield and quality.

[0003] Research on cotton GhSAC7 protein and its encoding gene can provide good genetic resources for exploring cotton fiber and cotton plant type. Summary of the Invention

[0004] This study discovered GhSAC7, a key gene regulating cotton fiber growth and development, and applied it to cotton variety improvement. The researchers cloned a cotton GhSAC7 protein-encoding gene (Gh_D03G0020; LOC107937826), with a coding sequence of 1791 nucleotides and a protein sequence of 596 amino acids. Protein expression and phenotypic analysis of gene-edited materials expressing the GhSAC7 gene revealed a phenotype characterized by slowed fiber initiation, reduced fiber density, and decreased total fiber volume. This suggests that GhSAC7 can effectively regulate cotton fiber development and promote fiber initiation. This provides a theoretical basis for cotton variety improvement and can be used to cultivate new cotton varieties with superior fiber quality.

[0005] Explanation of terms: Cotton fiber: The fiber covering the seeds of the Malvaceae plant is a seed fiber formed by the epidermal cells of the fertilized ovule through initiation, elongation, secondary cell wall thickening and fiber maturation. Unlike general bast fiber, its main component is cellulose, which is an important raw material for the textile industry.

[0006] The technical solution of the present invention is: In the first aspect, the present invention discloses cotton GhSAC7 protein, GhSAC7 protein encoding gene or containing GhSAC7 The application of a gene expression cassette or a recombinant plant expression vector in regulating the initial differentiation of cotton fibers, wherein the amino acid sequence of the cotton GhSAC7 protein is shown in SEQ ID No. 1.

[0007] Preferably, the nucleotide sequence of the gene encoding the cotton GhSAC7 protein is shown as SEQ ID No.2.

[0008] Preferably, the application is to increase the expression level and / or activity of the GhSAC7 protein or its encoding gene in the cotton, thereby promoting cotton fiber development and increasing the total fiber amount and fiber density.

[0009] Preferably, the application is to reduce the expression level and / or activity of the GhSAC7 protein or its encoding gene in the cotton, inhibit cotton fiber development, and reduce the total fiber amount and fiber density.

[0010] Second aspect: The present invention discloses a method for cultivating cotton that promotes cotton fiber development, increases the total fiber amount and fiber density, comprising the step of increasing the expression level and / or activity of the GhSAC7 protein in the recipient cotton; Preferably, the amino acid sequence of the GhSAC7 protein is shown in SEQ ID No. 1.

[0011] Preferably, the increasing of the expression level and / or activity of the GhSAC7 protein in the recipient cotton is achieved by introducing the gene encoding the GhSAC7 protein into the recipient cotton.

[0012] Preferably, the encoding gene of the GhSAC7 protein is introduced into the recipient cotton in the form of a recombinant vector.

[0013] Preferably, the nucleotide sequence of the gene encoding the cotton GhSAC7 protein is shown as SEQ ID No.2.

[0014] The advantages and positive effects of the present invention are: Through GhSAC7 The protein expression level and phenotype analysis of the gene editing materials were conducted, and the results showed that GhSAC7 The gene editing material has the phenotype of slowing down fiber initiation, reducing fiber density and reducing the total amount of fiber. GhSAC7 It can effectively regulate the development of cotton fibers, promote the initiation of cotton fibers, provide a theoretical basis for cotton variety improvement, and can be used to cultivate new cotton varieties with excellent fiber quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0016] Figure 1 The gene-edited cotton and wild-type cotton provided by the embodiment of the present invention are GhSAC7 genotype; Figure 2 is the protein expression level of GhSAC7 in the wild-type and gene-edited cotton provided in the embodiments of the present invention; Figure 3 The control group of cotton TM-1 and GhSAC7 Gene editing of cotton fiber initiation phenotypes; Figure 4 The control group cotton TM-1 and GhSAC7 Gene editing of mature fiber phenotype in cotton. DETAILED DESCRIPTION

[0017] Unless otherwise specified, the methods used in the following examples are all conventional methods.

[0018] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0019] Example 1 1. Protein GhSAC7 and its encoding gene ( Gh_D03G0020; LOC107937826 ) 1. cDNA is obtained RNA was extracted from ovules of upland cotton at day 0 of flowering and was reverse transcribed into cDNA.

[0020] 2. PCR amplification Using the cDNA obtained in step 1 as a template, primers F and R were used to obtain PCR products. The primer sequences are shown in Table 1: Table 1

[0021] 3. Sequencing of PCR products The PCR product obtained in step 2 was sequenced. The sequencing results showed that the PCR product contained a DNA fragment of 1791 bp in size. The nucleotide sequence of the DNA fragment was shown in Sequence 1. The gene shown in Sequence 1 was named GhSAC7 Gene. Gene GhSAC7 The encoded protein is named GhSAC7 protein. The GhSAC7 protein consists of 596 amino acids, and its amino acid sequence is shown in SEQ ID NO: 2.

[0022] SEQ ID No. 1: 1 mmeraescqk lytrlrlwef pdeyvieptd gssasslkin radasmklid aipecgsvrv61pkiqtifgvv gmlklvagsy liviterecv gsylghpifk vmslrilpcd hslktsppeq121kkvesefagl lkvaektcgl ffsydtnltl saqrlndlgd eskllplwrq aeprflwnny181mlevlidnkl dpyllpvvqg sfhnfqaaig keivditlia rrctrrngtr mwrrgadpdg241yvanfveteq ivqmngftas fvqvrgsmpf lweqivdlty kpkfeivkpe eaprvaerhf301ldlrkkygsv laidlvnttg gegrlsekfa savqpilsdd lryihfdfhk icghvhferl361silydqiadf ldkngyllln dkgekmkeql gvvrtncidc ldrtnvtqsm igrkmlelql421rrigvfaaee tisshtnlde kykilwanhg ddvsiqysgt palkgdfvry gkrtvqgiln481dfynalgryy fnnfsdgtkq daidllqghy ivsvsrdmtp psqkggleav ahfpaafclv541slglftils lsqarydlrh lffsalwati sigiaavvka ngrifcnrpr lhkprr。

[0023] SEQ ID No.2:

[0024] two, GhSAC7 Obtaining gene-edited cotton 1. Carry the correct sequencing GhSAC7 The intermediate vector of the gene was entrusted to Weimi Biotechnology Co., Ltd. to construct a gene editing vector for genetic transformation of cotton. The recipient cotton plant was TM-1, a genetically homozygous line of upland cotton, which originated from a breeding program in Texas, USA in the mid-20th century and was bred at the Texas Agricultural Experiment Station in the 1950s. It is a standard line of upland cotton. The T3 generation was finally obtained. GhSAC7 Gene-edited cotton.

[0025] 2. Identification of GhSAC7 gene-edited cotton The GhSAC7 gene-edited cotton was identified. The specific steps are as follows: Based on the DNA fragments near the target site of the gene-edited cotton, a pair of specific primers, GhSAC7D-CRISPR-F1 and GhSAC7D-CRISPR-R1, and GhSAC7D-CRISPR-F2 and GhSAC7D-CRISPR-R2, were designed (Table 2). Total DNA from the leaves of each gene-edited cotton line was extracted and used as a template for PCR amplification and sequencing to identify the genotype of the GhSAC7 gene-edited cotton. The results are shown in Figure 2. Figure 1 As shown, two gene-edited lines GhSAC7-cr1 and GhSAC7-cr2 Single-base insertions and deletions occurred at the target gene sites, resulting in gene frameshift and impaired protein function.

[0026] Table 2

[0027] 3. GhSAC7 Analysis of GhSAC7 expression levels in gene-edited cotton right GhSAC7 The protein expression levels of GhSAC7 in gene-edited cotton were analyzed. The specific steps were as follows: A peptide specific to the GhSAC7 protein was selected and submitted to GenScript Biotech Co., Ltd. for the preparation of a GhSAC7-specific antibody. Total protein from day 0 ovules of each GhSAC7 gene-edited cotton line was extracted. Western blotting was performed on these samples to determine the expression levels of GhSAC7 protein in each gene-edited line.

[0028] The results are as follows Figure 2 As shown, based on the expression level of GhSAC7 protein in the control cotton TM-1, the expression levels of GhSAC7 protein-edited cotton were lower than that of the wild type. Subsequent experiments were carried out using the above-mentioned GhSAC7 gene-edited cotton.

[0029] 4. GhSAC7 Analysis of fiber initiation phenotypes in gene-edited cotton Plant materials are as follows: control cotton TM-1, and gene-edited cotton GhSAC7-cr1 and GhSAC7-cr2 .

[0030] The control group cotton TM-1 and gene-edited cotton GhSAC7-cr1 and GhSAC7-cr2 The ovules at the same position of the cotton bolls on the 0th day after flowering were photographed using a scanning electron microscope to observe the cotton fiber phenotype.

[0031] The results are as follows Figure 3 As shown in the figure, it can be seen GhSAC7-cr1 and GhSAC7-cr2 The initial fiber density of the WT and WT were significantly lower than those of the wild type, indicating that GhSAC7 It can promote the initiation of cotton fibers and increase the density of cotton fibers.

[0032] 5. Analysis of mature fiber phenotypes of GhSAC7 gene-edited cotton Plant materials are as follows: control cotton TM-1, and GhSAC7 gene-edited cotton GhSAC7-cr1 and GhSAC7- cr2 .

[0033] The control group cotton TM-1 and GhSAC7 gene-edited cotton were selected GhSAC7-cr1 and GhSAC7-cr2 Observe the cotton fiber phenotype of seeds at the same position of the cotton boll on the plant; and count the fiber length and the number of fibers on a seed.

[0034] The results are as follows Figure 4 As shown in the figure, gene-edited cotton GhSAC7-cr1 and GhSAC7-cr2 The fiber density of the WT and wild type were significantly lower than those of the wild type. GhSAC7 It can promote the development of cotton fibers and increase the total fiber amount and fiber density.

[0035] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Cotton GhSAC7 protein, GhSAC7 protein encoding gene or containing GhSAC7 The application of a gene expression cassette or a recombinant plant expression vector in regulating the initial differentiation of cotton fibers, wherein the amino acid sequence of the cotton GhSAC7 protein is shown in SEQ ID No.

1.

2. The use according to claim 1, characterized in that The nucleotide sequence of the GhSAC7 protein encoding gene is shown in SEQ ID No.

2.

3. The use according to claim 2, characterized in that The application is to increase the expression level and / or activity of the GhSAC7 protein or its encoding gene in the cotton, promote cotton fiber development, and increase the total fiber amount and fiber density.

4. The use according to claim 2, characterized in that The application is to reduce the expression level and / or activity of the GhSAC7 protein or its encoding gene in the cotton, inhibit cotton fiber development, and reduce the total fiber amount and fiber density.

5. A method for cultivating cotton that promotes cotton fiber development and increases the total fiber amount and fiber density, comprising the step of increasing the expression level and / or activity of GhSAC7 protein in recipient cotton.

6. The method according to claim 5, wherein The amino acid sequence of the GhSAC7 protein is shown in SEQ ID No.

1.

7. The method according to claim 5, wherein The increasing of the expression level and / or activity of the GhSAC7 protein in the recipient cotton is achieved by introducing the coding gene of the GhSAC7 protein into the recipient cotton.

8. The method according to claim 7, wherein The coding gene of the GhSAC7 protein is introduced into the recipient cotton in the form of a recombinant vector.

9. The method according to claim 8, wherein The nucleotide sequence of the gene encoding the cotton GhSAC7 protein is shown in SEQ ID No.2.

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