Cotton GhSNL1 protein and its encoding gene and application

Abstract

The present invention discloses cotton GhSNL1 protein and its coding gene and application. The cotton GhSNL1 protein coding gene (Gh_A01G1757; LOC107925613) has a coding sequence of 1290 nucleotides and a protein sequence of 429 amino acids. GhSNL1 Phenotypic analysis of CRISPR‑Cas9 gene editing materials revealed GhSNL1 The knockout material of the gene showed the phenotype of slow fiber initiation, shortened fiber density and regeneration of bolls in mature plants, indicating that GhSNL1 The gene is involved in the development of cotton fibers, promotes the initiation of cotton fibers and affects plant morphology, providing a theoretical basis for cotton variety improvement and can be used to breed new cotton varieties with excellent fiber quality and high yield.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, and in particular to cotton GhSNL1 protein, its encoding gene and application. Background Art

[0002] cotton( Gossypium spp. ) As an economic crop widely planted around the world, its cotton fiber is an important source of natural fiber in textiles. Cotton fiber is the fiber covering its seeds, which develops from the epidermal cells of the fertilized ovules. The cotton fiber development process is divided into four stages: initiation, elongation, secondary cell wall thickening and fiber maturation. Among them, the initiation and elongation stages determine the number and length of cotton fibers. Therefore, the revelation of the cotton fiber development process, especially the regulatory mechanism of the initiation and elongation stages, has important practical significance for improving cotton yield and quality. At the same time, the plant type of cotton is closely related to the boll setting rate. The angle of the fruit branch, the aspect ratio of the plant (the ratio of the longitudinal length of the plant to the lateral width) and the boll setting rate are all significantly positively correlated. The smaller the fruit branch, or the larger the aspect ratio of the plant, the higher the boll setting rate.

[0003] Research on cotton GhSNL1 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 an important gene that regulates cotton fiber growth and development GhSNL1 The cotton GhSNL1 protein coding gene (Gh_A01G1757; LOC107925613) has a coding sequence of 1290 nucleotides and a protein sequence of 429 amino acids. GhSNL1 Phenotypic analysis of CRISPR-Cas9 gene editing materials revealed GhSNL1 The knockout material of the gene showed the phenotype of slowed fiber initiation, shortened fiber density and regeneration of bolls in mature plants. GhSNL1 The gene is involved in the development of cotton fibers, promotes the initiation of cotton fibers and affects plant morphology, providing a theoretical basis for cotton variety improvement and can be used to breed new cotton varieties with excellent fiber quality and high yield.

[0005] Explanation of terms:

[0006] Cotton fiber: Cotton fiber, the fiber covering the seeds of the Malvaceae plant, is formed by the epidermal cells of the fertilized ovule through initiation, elongation, secondary cell wall thickening, and fiber maturation. Unlike conventional bast fibers, its primary component is cellulose. It is an important raw material for the textile industry.

[0007] Zero fruit branch type: The fruit branches are not promoted to grow, there are no fruit nodes, and the cotton bolls are directly attached to the axils of the main stem.

[0008] Type 2 fruit branch type: It has multiple fruit branches and may continue to extend and add nodes, also known as infinite fruit branch type.

[0009] The technical solution of the present invention is:

[0010] In the first aspect, the present invention discloses cotton GhSNL1 protein, GhSNL1 protein encoding gene or containing GhSNL1 The application of the gene expression cassette or recombinant plant expression vector in regulating cotton fiber growth and development and plant morphology, the amino acid sequence of the cotton GhSNL1 protein is shown in SEQ ID No.1.

[0011] Preferably, the nucleotide sequence of the gene encoding the cotton GhSNL1 protein is shown as SEQ ID No.2.

[0012] Preferably, the application is to increase the expression level and / or activity of the GhSNL1 protein or its encoding gene in the cotton, thereby increasing the cotton fiber density and length, and the cotton plant type is a zero-fruit branch type.

[0013] Preferably, the application is to reduce the expression level and / or activity of the GhSNL1 protein or its encoding gene in the cotton, so that the cotton fiber density is reduced, the cotton fiber length is shortened, and the cotton plant type is a dimorphic fruit branch type.

[0014] Second aspect: The present invention discloses a method for cultivating cotton with increased cotton fiber density, increased cotton fiber length, and a zero-type fruiting branch type cotton plant, comprising the step of increasing the expression level and / or activity of the GhSNL1 protein in the recipient cotton;

[0015] Preferably, the amino acid sequence of the GhSNL1 protein is the amino acid sequence of cotton GhSNL1 protein as shown in SEQ ID No. 1.

[0016] A method for cultivating cotton with reduced cotton fiber density, shortened cotton fiber length, and a two-type fruiting branch type cotton plant, comprising the step of increasing the expression level and / or activity of GhSNL1 protein in recipient cotton;

[0017] The amino acid sequence of the GhSNL1 protein is the amino acid sequence of cotton GhSNL1 protein as shown in SEQ ID No. 1.

[0018] Preferably, the increasing the expression level and / or activity of the GhSNL1 protein in the recipient cotton is achieved by introducing a gene encoding the GhSNL1 protein into the recipient cotton.

[0019] Preferably, the gene encoding the GhSNL1 protein is introduced into the recipient cotton in the form of a recombinant vector.

[0020] Preferably, reducing the expression level and / or activity of the GhSNL1 protein in the recipient cotton is achieved by knocking out or inhibiting the expression of the gene encoding the GhSNL1 protein in the recipient cotton.

[0021] Preferably, the nucleotide sequence of the gene encoding the cotton GhSNL1 protein is shown as SEQ ID No.2.

[0022] The advantages and positive effects of the present invention are:

[0023] The present invention is through GhSNL1 Phenotypic analysis of CRISPR-Cas9 gene editing materials revealed GhSNL1 The knockout material of the gene showed the phenotype of slowed fiber initiation, shortened fiber density and regeneration of bolls in mature plants. GhSNL1 The gene is involved in the development of cotton fibers, promotes the initiation of cotton fibers and affects plant morphology, providing a theoretical basis for cotton variety improvement and can be used to breed new cotton varieties with excellent fiber quality and high yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] 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.

[0025] Figure 1 The invention provides a method for identifying the transgenic plant at the DNA level. GhSNL1 Plant materials positive for gene editing vectors;

[0026] Figure 2 The DNA level detection provided by the embodiment of the present invention GhSNL1-m1 and GhSNL1-m2 Gene editing sequences;

[0027] Figure 3 It is the gene editing material provided by the embodiment of the present invention GhSNL1-m1, GhSNL1-m2 and wild-type cotton fiber initiation phenotype;

[0028] Figure 4 It is the gene editing material provided by the embodiment of the present invention GhSNL1-m1, GhSNL1-m2 and wild-type cotton mature fiber length phenotype;

[0029] Figure 5 It is the gene editing material provided by the embodiment of the present invention GhSNL1-m1, GhSNL1-m2 and wild-type cotton boll-forming phenotype. DETAILED DESCRIPTION

[0030] Unless otherwise specified, the methods used in the following examples are all conventional methods.

[0031] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0032] Example 1

[0033] 1. Obtaining the protein GhSNL1 and its encoding gene (Gh_A01G1757; LOC107925613)

[0034] 1. cDNA is obtained

[0035] RNA was extracted from ovules of upland cotton at -2, 0 and 2 days after anthesis and reverse transcribed into cDNA.

[0036] 2. PCR amplification

[0037] Using the cDNA mixture 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:

[0038] Table 1

[0039]

[0040] 3. Sequencing of PCR products

[0041] The PCR product obtained in step 2 was sequenced. The sequencing results showed that the PCR product contained a DNA fragment of 1290 bp in size. The nucleotide sequence of the DNA fragment was shown in SEQ ID No. 2. The gene shown in SEQ ID No. 2 was named GhSNL1 Gene. Gene GhSNL1 The encoded protein is named GhSNL1 protein. The GhSNL1 protein consists of 429 amino acids, and its amino acid sequence is shown in SEQ ID No.1.

[0042] SEQ ID No. 1:

[0043] MDASDLSFRKSKMEAFLERVKEHMKADQYTLLLISVDEFNNGGITVYRLNEIVEVLLREYPGFFTQFQFVLNFANGVTRRVQSADSSNKGKRKLASDEDGEIDGLNETKDQLAEAMEFCEKVMKQTSYDKYLDLLKHLYSYGTGKIIMVDLKTAIAENFQALGEDFHLFEFYTNISRPTSSSSSSSESEGKKRNQEVDIVKESCKPRQKGPSLESKMSNKKKKPKEEIEKVRKSYKVKREGPSSSSLTKPKKEINKPEEEELEKVTESYYLLPENLSGVHSTEIDEIGKQVLNFSTFSKGVYNTNKQKGPEITKQEMVMNRKEDEMFVMDMQMEWLRSTKKNAMKLFQDISERKIKEPTMADVDEYFTSSNYRYLVKMYNESGPWLVDRLRHAKPTILPVIIKRLKQKDIVPYRELCQQHQQMLEDQQ.

[0044] SEQ ID No.2:

[0045]

[0046] two, GhSNL1 Obtaining gene-edited cotton

[0047] 1. In GhSNL1 Two editing targets suitable for the CRISPR-Cas9 system were designed (Table 2) and commissioned by Weimi Biotechnology Co., Ltd. to be constructed into 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 bred at the Texas Agricultural Experiment Station in the 1950s. As a genetically homozygous line of Upland cotton, it is a standard line of Upland cotton). The final T1 generation was obtained. GhSNL1 Gene-edited cotton.

[0048] Table 2

[0049]

[0050] 2. GhSNL1 Identification and propagation of gene-edited cotton

[0051] The transgenic cotton identified as T1 generation was genotyped. The specific steps were as follows: two pairs of primers were designed to identify the inserted fragments in the transgenic cotton, CAS9-F1 and CAS9-R1, and CAS9-F2 and CAS9-R2 (Table 3). Total DNA from the leaves of each transgenic cotton line was extracted and used as a template for PCR amplification and identification. GhSNL1 Positive seedlings of gene-edited cotton.

[0052] Further in GhSNL1 Primers GhSNL1-JD-F and GhSNL1-JD-R were designed to amplify the upstream and downstream targets of the gene (Table 3). PCR amplification was performed using total leaf DNA as a template, and the products were sequenced by next-generation sequencing. Successfully edited cotton lines were propagated, and total leaf DNA was extracted and used as a template for PCR amplification in the next generation. The products were sequenced by next-generation sequencing. Homozygous lines were ligated to a T-vector for next-generation sequencing to confirm their genotypes.

[0053] Table 3

[0054]

[0055] The results are as follows Figure 1 As shown in the figure, GhSNL1 Two strains of gene-edited cotton GhSNL1-m1 and GhSNL1-m2 The target fragment on the inserted fragment can be amplified. Figure 2 As can be seen, gene-edited cotton lines GhSNL1-m1The target site 1 deletion resulted in 3 nucleotides, and the target site 2 deletion resulted in 31 nucleotides, causing a frameshift mutation. GhSNL1-m2 The deletion of 42 nucleotides (i.e., 14 amino acids) starts from the upstream of target site 1, and then a single base A is inserted at target site 2 to cause a frameshift mutation.

[0056] 3. GhSNL1 Analysis of fiber initiation phenotypes in gene-edited cotton

[0057] Plant materials are as follows: wild-type cotton TM-1, and GhSNL1 Two homozygous lines of gene-edited cotton GhSNL1- m1 and .

[0058] Select wild-type TM-1 and GhSNL1-m2 and GhSNL1-m1 The ovules at the same position of the 0 DPA cotton boll were photographed using a scanning electron microscope to observe the cotton fiber phenotype.

[0059] The results are as follows GhSNL1-m2 As shown in the figure, it can be seen Figure 3 and GhSNL1-m1 The fiber density of the mutants was significantly lower than that of the wild type, indicating that GhSNL1 can promote cotton fiber development and increase cotton fiber density.

[0060] 4. GhSNL1-m2 Phenotypic analysis of mature fiber length in gene-edited cotton

[0061] Plant materials are as follows: wild-type cotton TM-1 and two homozygous lines of GhSNL1 gene-edited cotton GhSNL1 m1 and GhSNL1- .

[0062] Select wild-type TM-1 and and GhSNL1-m2 The seeds were taken from the same position of the cotton bolls on the cotton plants to observe the cotton fiber phenotype and count the cotton fiber length.

[0063] The results are as follows GhSNL1-m1 As shown in the figure, it can be seen that the fiber lengths of GhSNL1-m1 and GhSNL1-m2 are significantly lower than those of the wild type, indicating that GhSNL1 can promote cotton fiber development and increase cotton fiber density.

[0064] 5. GhSNL1-m2 Plant type analysis of gene-edited cotton

[0065] The plant materials are as follows: wild-type cotton TM-1 and two homozygous lines of GhSNL1 gene-edited cotton GhSNL1-m1 and GhSNL1-m2.

[0066] Wild-type TM-1, GhSNL1-m1 and GhSNL1-m2 cotton plants with the same growth period were selected to observe the cotton growth phenotype.

[0067] The results are as follows Figure 4 As shown, the wild type TM-1 is a dimorphic fruit branch type, while the mutant GhSNL1 and Figure 5 GhSNL1-m1 GhSNL1-m2 The results of the boll count showed that the wild-type TM-1 had an average of 13.375 bolls per plant, while the mutants GhSNL1-m1 and GhSNL1-m2 had 23.125 and 16.125 bolls per plant, respectively.

[0068] 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. Application of knocking out the cotton GhSNL1 gene in regulating cotton fiber growth and development and cotton plant morphology, wherein knocking out the cotton GhSNL1 gene reduces cotton fiber density, shortens cotton fiber length, and produces a zero-type fruiting branch type cotton plant. The nucleotide sequence of the GhSNL1 gene is shown in SEQ ID No.

2.

2. The use according to claim 1, characterized in that The amino acid sequence of the protein encoded by the GhSNL1 gene is shown in SEQ ID No.

1.

3. A method for cultivating cotton with reduced cotton fiber density, shortened cotton fiber length, and a zero-type fruiting branch type cotton plant shape, characterized in that: The cotton GhSNL1 gene is knocked out, and the nucleotide sequence of the GhSNL1 gene is shown in SEQ ID No. 2.

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