Application of cotton GhSAC7 protein in regulating initiation of cotton fiber differentiation

Abstract

The application discloses a cotton GhSAC7 protein coding gene (Gh_D03G0020; LOC107937826), the coding sequence is 1791 nucleotides, and the protein sequence is 596 amino acids. GhSAC7 The protein expression level analysis and phenotype analysis are carried out on the gene editing material of the gene, and the results show that GhSAC7 The gene editing material of the gene has the phenotypes of slow fiber initiation, reduced fiber density and reduced total fiber amount. GhSAC7 The application can effectively regulate cotton fiber development, promote the initiation of cotton fiber, provide a theoretical basis for cotton variety improvement, and be used for cultivating new cotton varieties with excellent fiber quality.

Description

TECHNICAL FIELD

[0001] The application relates to the field of genetic engineering, in particular to application of cotton GhSAC7 protein in regulation of cotton fiber initial differentiation. BACKGROUND

[0002] Cotton (Gossypium spp.) is one of the most important economic crops in the world, and its seed fiber (cotton fiber) is an important raw material for the textile industry. The development process of cotton fiber 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, so the research on the molecular mechanism of the initiation and elongation stages of cotton fibers has important practical significance for the improvement of cotton yield and quality.

[0003] Research and exploration of cotton GhSAC7 protein and its coding gene can provide good gene resources for the study of cotton fiber and cotton plant type. SUMMARY

[0004] The application finds that the important gene GhSAC7 regulating the growth and development of cotton fiber is applied to cotton variety improvement. The application clones a cotton GhSAC7 protein coding gene (Gh_D03G0020; LOC107937826), the coding sequence is 1791 nucleotides, and the protein sequence is 596 amino acids. Through protein expression level analysis and phenotype analysis of the gene editing material of the GhSAC7 gene, it is found that the gene editing material of the GhSAC7 gene has the phenotypes of slow fiber initiation, reduced fiber density and reduced total fiber amount. Accordingly, it is indicated that GhSAC7 can effectively regulate cotton fiber development and promote the initiation of cotton fiber. The application provides a theoretical basis for cotton variety improvement and can be used for cultivating new cotton varieties with excellent fiber quality.

[0005] TERMS EXPLANATION:

[0006] Cotton fiber: the fiber covered on the seed of Malvaceae cotton plant, which is a seed fiber formed by the epidermal cells of fertilized ovule through initiation, elongation, secondary cell wall thickening and fiber maturation, and is different from general phloem fiber, and the main component of the fiber is cellulose, which is an important raw material for the textile industry.

[0007] The technical scheme of the application is as follows:

[0008] The first aspect: the application discloses application of cotton GhSAC7 protein, GhSAC7 protein coding gene or expression cassette containing GhSAC7 The amino acid sequence of the cotton GhSAC7 protein is shown as SEQ ID No. 1.

[0009] Preferably, the nucleotide sequence of the gene encoding the cotton GhSAC7 protein is shown in SEQ ID No. 2.

[0010] Preferably, the application is to increase the expression level and / or activity of GhSAC7 protein or its encoding gene in the cotton, thereby promoting cotton fiber development and increasing the total fiber content and fiber density.

[0011] Preferably, the application is to reduce the expression level and / or activity of GhSAC7 protein or its encoding gene in the cotton, thereby inhibiting cotton fiber development and reducing the total fiber volume and fiber density.

[0012] The second aspect: This invention discloses a method for cultivating cotton that promotes cotton fiber development and increases total fiber content and fiber density, including the step of increasing the expression level and / or activity of GhSAC7 protein in recipient cotton;

[0013] Preferably, the amino acid sequence of the GhSAC7 protein is shown in SEQ ID No. 1.

[0014] Preferably, the increase in the expression level and / or activity of GhSAC7 protein in the recipient cotton is achieved by introducing the gene encoding the GhSAC7 protein into the recipient cotton.

[0015] Preferably, the gene encoding the GhSAC7 protein is introduced into the recipient cotton in the form of a recombinant vector.

[0016] Preferably, the nucleotide sequence of the gene encoding the cotton GhSAC7 protein is shown in SEQ ID No. 2.

[0017] The advantages and positive effects of this invention are as follows:

[0018] Through the GhSAC7 Protein expression level analysis and phenotypic analysis were performed on gene-edited materials, and the results showed that... GhSAC7 Gene-edited materials exhibit a phenotype characterized by slowed fiber initiation, reduced fiber density, and decreased total fiber volume. This indicates that... GhSAC7 It can effectively regulate cotton fiber development, 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. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show the embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The gene-edited cotton and wild-type cotton provided in the embodiments of the present invention GhSAC7 genotype;

[0021] Figure 2 This refers to the protein expression levels of GhSAC7 in wild-type and gene-edited cotton provided in the embodiments of the present invention;

[0022] Figure 3 The control group cotton TM-1 provided in this embodiment of the invention and GhSAC7 Gene-edited cotton fiber initiation phenotype;

[0023] Figure 4 The control group cotton TM-1 provided in the embodiments of the present invention and GhSAC7 Phenotype of mature cotton fibers edited with genes. Detailed Implementation

[0024] Unless otherwise specified, the methods used in the following embodiments are conventional methods.

[0025] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0026] Example 1

[0027] I. Protein GhSAC7 and its encoding gene ( Gh_D03G0020; LOC107937826 The acquisition of )

[0028] 1. Obtaining cDNA

[0029] RNA was extracted from ovules of upland cotton at day 0 of flowering and reverse transcribed into cDNA.

[0030] 2. PCR amplification

[0031] 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.

[0032] Table 1

[0033]

[0034] 3. Sequencing of PCR products

[0035] The PCR product obtained in step 2 was sequenced. Sequencing results showed that the PCR product contained a 1791 bp DNA fragment, the nucleotide sequence of which is shown in Sequence 1. The gene shown in Sequence 1 was named... GhSAC7 Genes. GhSAC7The encoded protein is named GhSAC7 protein. GhSAC7 protein consists of 596 amino acids, and its amino acid sequence is shown in Sequence 2.

[0036] SEQ ID No. 1:

[0037] 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 ahfpaafclv541slglfftils lsqarydlrh lffsalwati sigiaavvka ngrifcnrpr lhkprr.

[0038] SEQ ID No. 2:

[0039]

[0040] two, GhSAC7 The acquisition of gene-edited cotton

[0041] 1. Carry the correctly sequenced... GhSAC7 The intermediate gene vector was entrusted to Weimi Biotechnology Co., Ltd. to construct a gene-editing vector for genetic transformation of cotton. The recipient cotton plant was upland cotton TM-1 (originating from a breeding program in Texas, USA in the mid-20th century, and selected at the Texas Agricultural Experiment Station in the 1950s; it is a genetically homozygous line of upland cotton and a standard line of upland cotton). The final result was the T3 generation. GhSAC7 Gene-edited cotton.

[0042] 2. Identification of GhSAC7 gene-edited cotton

[0043] The GhSAC7 gene-edited cotton was identified. The specific steps are as follows: Based on the DNA fragment near the target site in the gene-edited cotton, a pair of specific primers, GhSAC7D-CRISPR-F1 and GhSAC7D-CRISPR-R1, and GhSAC7D-CRISPR-F2 and GhSAC7D-CRISPR-R2 (Table 2), were designed. Total DNA was extracted from the leaves of each gene-edited cotton line. Using this DNA as a template, PCR amplification and sequencing were performed to identify the genotype of the GhSAC7 gene-edited cotton. The results are as follows: Figure 1 As shown, two gene-edited lines GhSAC7-cr1 and GhSAC7-cr2 The target gene sites produced single-base insertions and deletions, resulting in gene frameshifting and impaired protein function.

[0044] Table 2

[0045]

[0046] 3. GhSAC7 Analysis of GhSAC7 expression levels in gene-edited cotton

[0047] right GhSAC7 The protein expression level of GhSAC7 in gene-edited cotton was analyzed. The specific steps were as follows: A specific peptide of GhSAC7 protein was selected, and a GhSAC7-specific antibody was prepared by GenScript Biotech Co., Ltd. Total protein was extracted from day 0 ovules of each GhSAC7 gene-edited cotton line. Using these samples, Western blotting was performed to detect the expression level of GhSAC7 protein in each gene-edited line.

[0048] The results are as follows Figure 2As shown, compared with the expression level of GhSAC7 protein in the control group cotton TM-1, the expression level of GhSAC7 protein in the edited cotton was lower than that in the wild type. The cotton edited with the above GhSAC7 gene was used for subsequent experiments.

[0049] 4. GhSAC7 Fiber initiation phenotype analysis of gene-edited cotton

[0050] The plant materials are as follows: control group cotton TM-1 and gene-edited cotton. GhSAC7-cr1 and GhSAC7-cr2 .

[0051] The control group cotton TM-1 and gene-edited cotton were selected. GhSAC7-cr1 and GhSAC7-cr2 Ovules at the same position on the cotton boll 0 days after flowering were observed using scanning electron microscopy to examine the phenotype of cotton fibers.

[0052] The results are as follows Figure 3 As shown in the figure, it can be seen that... GhSAC7-cr1 and GhSAC7-cr2 The initial fiber density of the filaments was significantly lower than that of the wild type, indicating that... GhSAC7 It can promote the initiation of cotton fibers and increase the density of cotton fibers.

[0053] 5. Phenotypic analysis of mature fibers from GhSAC7 gene-edited cotton

[0054] The plant materials are as follows: control group cotton TM-1 and GhSAC7 gene-edited cotton. GhSAC7-cr1 and GhSAC7- cr2 .

[0055] cotton TM-1 (control group) and GhSAC7 gene-edited cotton were selected. GhSAC7-cr1 and GhSAC7-cr2 Observe the cotton fiber phenotype of seeds from the same position on the cotton boll; and count the fiber length and the number of fibers on a single seed.

[0056] The results are as follows Figure 4 As shown in the figure, gene-edited cotton can be seen. GhSAC7-cr1 and GhSAC7-cr2 The fiber density of the two types was significantly lower than that of the wild type, indicating that... GhSAC7 It can promote the development of cotton fibers and increase the total amount of fibers and fiber density.

[0057] 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 within the protection scope of the present invention.

Claims

1. The application of the GhSAC7 protein-encoding gene in regulating cotton fiber initiation differentiation, characterized in that, The nucleotide sequence of the GhSAC7 protein encoding gene is shown in SEQ ID No.

2. The application is to increase the expression level and / or activity of GhSAC7 protein or its encoding gene in cotton, thereby promoting cotton fiber development and increasing the total fiber content and fiber density.

2. The application as described in claim 1, characterized in that, The amino acid sequence of the protein encoded by the GhSAC7 protein-coding gene is shown in SEQ ID No.

1.

3. A method for cultivating cotton that promotes fiber development and increases total fiber content and fiber density, characterized in that, The procedure includes steps to increase the expression level and / or activity of the GhSAC7 protein in the recipient cotton, the nucleotide sequence of the GhSAC7 protein encoding gene being shown in SEQ ID No. 2.

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