CN112980876A

CN112980876A - Application of GhGPAT12 protein and GhGPAT25 protein in regulation and control of cotton male reproductive development

Info

Publication number: CN112980876A
Application number: CN202110269686.4A
Authority: CN
Inventors: 魏恒玲; 张蒙; 喻树迅; 王寒涛; 马亮; 芦建华; 喻晓云
Original assignee: Institute of Cotton Research of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Cotton Research of Chinese Academy of Agricultural Sciences
Priority date: 2021-03-12
Filing date: 2021-03-12
Publication date: 2021-06-18
Anticipated expiration: 2041-03-12
Also published as: CN112980876B

Abstract

The invention provides application of GhGPAT12 protein and GhGPAT25 protein in regulation and control of cotton male reproductive development, and relates to the technical field of plant genetic engineering. The amino acid sequence of the GhGPAT12 protein is shown in SEQ ID No. 1; the amino acid sequence of the GhGPAT25 protein is shown in SEQ ID No. 3. The invention discloses that 2 glycerol-3-phosphate acyltransferase proteins, namely GhGPAT12 protein and GhGPAT25, play a role in regulating and controlling cotton male reproductive development in a function redundancy mode, and provide breeding resources for cultivating cotton male sterile lines.

Description

Application of GhGPAT12 protein and GhGPAT25 protein in regulation and control of cotton male reproductive development

Technical Field

The invention relates to the technical field of plant genes, in particular to application of GhGPAT12 protein and GhGPAT25 protein in regulation and control of male reproductive development of cotton.

Background

Cotton plays an important role in agricultural production as one of important fiber crops. Cotton has significant heterosis. The utilization of cotton hybrid vigor still mainly adopts manual castration, which consumes a great deal of labor force and has high seed production cost. The male sterile line refers to male sterility caused by the defect of the development of plant stamens. The cultivation of the male sterile line cotton has important significance for cotton crossbreeding.

Anthers, which are the site of pollen formation and development, are the core part of the entire stamen. The surface of the anther is covered with a layer of fatty structure, which becomes the stratum corneum. The stratum corneum is generally composed of cutin (cutin), cornified components (cutin), and epidermal wax (cuticular wax). The coverage of the stratum corneum provides a protective barrier to the formation and development of anthers and pollen. In the process of pollen development, tapetum provides a precondition for the morphogenesis and development of microspores. The tapetum provides a lipid-like raw material for the exine formation of the microspores, providing nutrients and contents for the maturation of the pollen grains. In these processes, the tapetum is subjected to a degradation process of programmed cell death, and the degraded tapetum residue is enriched in pollen grains indicating the formation of an oil-containing layer. Based on previous researches on plants such as arabidopsis thaliana and rice, abnormal degradation (early or delayed) of the tapetum is found to cause abnormal pollen development, thereby causing male sterility.

The pollen wall is a complex multicellular layer on the pollen surface and is essential for the completion of the basic morphology of plant anthers. The pollen wall mainly comprises an outer wall (Exine) and an inner wall (intein) which contain an oil layer (tryptine). The pollen outer wall is generally divided into an outer wall outer layer (seine) and an outer wall inner layer (Nexine) which contain a canopy (Tectum) and a pillar (bactula). The main components of the pollen outer wall are sporopollenin. The main synthetic components of sporopollenin are phenols (Phenolics), Fatty Acids (fat Acids) and Alkanes (Alkanes), and the stable physicochemical properties of sporonin are formed by polymerization of the phenols, the Fatty Acids and the Alkanes (Alkanes), so that pollen walls have strong resistance and protection capability.

Glycerol-3-phosphate acyltransferase (GPAT) is the rate-limiting enzyme for Triacylglycerol (TAG) biosynthesis, catalyzing the initiation step of TAG biosynthesis. GPATs are primarily responsible for the transfer of fatty acyl groups from acyl carrier protein (acyl-ACP) or acyl coenzyme A (acyl-CoA) to the sn-1 or sn-2 position of Glycerol-3-phosphate (Glycerol-3-phosphate, G3P). It has been found that sn-1-GPAT is generally involved in the synthesis of storage lipids, such as ATS1 and AtGPAT9 of Arabidopsis thaliana; sn-2-GPAT is involved in the formation of some polyesters (polyesters) such as cutin (cutin) and suberin (suberin), such as AtGPAT1-8 of Arabidopsis thaliana.

Currently, it is not clear whether glycerol-3-phosphate acyltransferase-related genes have an effect on the normal development of anthers and pollen in cotton plants. In addition, few studies have been reported on genes regulating male sterility in cotton.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide application of GhGPAT12 protein and GhGPAT25 protein in regulation and control of cotton male reproductive development. The invention discloses that 2 glycerol-3-phosphate acyltransferase proteins, namely GhGPAT12 protein and GhGPAT25, play a role in regulating and controlling cotton male reproductive development in a function redundancy mode, and provide breeding resources for cultivating cotton male sterile lines.

The technical scheme provided by the invention is as follows:

the invention provides application of GhGPAT12 protein and GhGPAT25 protein in regulation and control of cotton male reproductive development, wherein the amino acid sequence of the GhGPAT12 protein is shown as SEQ ID No. 1; the amino acid sequence of the GhGPAT25 protein is shown in SEQ ID No. 3.

The invention also provides application of GhGPAT12 protein and GhGPAT25 protein in regulation and control of cotton pollen development, wherein the amino acid sequence of the GhGPAT12 protein is shown as SEQ ID No. 1; the amino acid sequence of the GhGPAT25 protein is shown in SEQ ID No. 3.

In the scheme of the invention, the GhGPAT12 protein and the GhGPAT25 protein have a role in the development of pollen and anthers of cotton.

In a specific embodiment, the amino acid sequence may be substituted, deleted or added with one or more amino acid residues to derive an amino acid sequence with equivalent functions.

The invention also provides application of GhGPAT12 protein and GhGPAT25 protein in breeding male sterile transgenic cotton, wherein the amino acid sequence of the GhGPAT12 protein is shown as SEQ ID No. 1; the amino acid sequence of the GhGPAT25 protein is shown in SEQ ID No. 3.

GhGPAT12 and GhGPAT25 are a pair of paralogous genes, and the invention discovers that the pair of genes are mainly expressed in the tetrad stage of microspores by fluorescence quantification. But also have similar expression patterns. Then, the pair of genes is knocked out through CRISPR/Cas9, and the completely edited transgenic plant is found to be completely male sterile, while a single plant containing GhGPAT12 or/and GhGPAT25 wild-type sequences is normally fertile, which indicates that GhGPAT12 and GhGPAT25 are important in cotton anther development and have functional redundancy. Therefore, the function of the protein or gene can be utilized in breeding male sterile transgenic cotton.

The invention also provides application of the coding genes of GhGPAT12 protein and GhGPAT25 protein or biological materials containing the coding genes in regulation and control of cotton male reproductive development, pollen development or cultivation of male sterile transgenic cotton, wherein the CDS sequence of the coding gene of the GhGPAT12 protein is shown as SEQ ID No. 2; the CDS sequence of the coding gene of the GhGPAT25 protein is shown as SEQ ID No. 4.

It will be understood by those skilled in the art that, in the case where the amino acid sequences of the GhGPAT12 protein and the GhGPAT25 protein are known, different nucleotide sequences encoding genes encoding proteins having the same functions as the GhGPAT12 protein or the GhGPAT25 protein can be obtained according to the degeneracy of codons.

In a specific embodiment, the biological material comprises an expression cassette, an expression vector or a host cell.

In one embodiment, the use is by inactivating the genes encoding the GhGPAT12 protein and the GhGPAT25 protein, or by reducing or inhibiting the expression of the genes encoding the GhGPAT12 protein and the GhGPAT25 protein.

In one embodiment, the application comprises the steps of inactivating coding genes of GhGPAT12 protein and GhGPAT25 protein by adopting a gene knockout, gene knock-down or gene editing method, or reducing or inhibiting the expression of the coding genes of GhGPAT12 protein and GhGPAT25 protein, so that the mutant cotton has a male sterility character.

In one embodiment, the application achieves inhibiting the expression of the gene or loss of function of the gene by CRISPR/Cas9 system, TALEN system, zinc finger enzyme system, RNA interference technology.

In one embodiment, the application adopts CRISPR/Cas9 system construction vector to carry out gene editing of coding genes of GhGPAT12 protein and GhGPAT25 protein;

preferably, the target site sequence of the designed CRISPR/Cas9 vector is shown as SEQ ID No. 5 and SEQ ID No. 6.

The invention also provides a method for cultivating male sterile line transgenic cotton, which comprises the step of obtaining a male sterile line transgenic plant by inactivating coding genes of GhGPAT12 protein and GhGPAT25 protein or reducing or inhibiting the expression of the coding genes of GhGPAT12 protein and GhGPAT25 protein.

In one embodiment, the cotton comprises gossypium barbadense and gossypium hirsutum, preferably gossypium hirsutum.

Has the advantages that:

(1) the invention identifies the functions of the GhGPAT12 protein and the GhGPAT25 protein of the cotton plants in regulating and controlling pollen development for the first time, provides the application of the GhGPAT12 protein and the GhGPAT25 protein in controlling the male reproductive development of cotton, and provides an effective way for the genetic modification of the cotton plants.

(2) The invention provides a novel method for cultivating cotton male sterile materials by knocking out or mutating GhGPAT12 and GhGPAT25 genes.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is an expression pattern diagram of GhGPAT12/25 (wherein, MMC: pollen mother cell period, MC: pollen mother cell meiosis period, TTP: tetrad period, eUNP: early mononuclear period, lUNP: late mononuclear period, BNP: binuclear period, MP: maturation period, stamen: stamen, root: root, stem, leaf: leaf, petal: petal, pistil: pistil, torus: receptacle, calyx: para);

FIG. 2 shows the PCR detection results of cotton transgenic seedlings according to the present invention (the numbers of the lines of the samples to be detected in lanes from left to right in the gel diagram are 6-1, 6-2, 6-6, 6-7, 6-9, 6-11, 6-12, 6-15, 6-20, 6-23, 6-25, 6-26, 6-28, 6-29, and 6-30, in which B represents a blank control, N represents a negative control, and P represents a positive control);

FIG. 3 is a schematic diagram of editing information of transgenic plants of different editing types and GhGPAT12/25 gene CRISPR/Cas9 knockout T₂Representing the plant phenotype chart.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Experimental materials:

the cotton material selected in the experiment is HM-1, the cotton material is planted in a key laboratory test field (Anyang white wall) of the national institute of Cotton biology of the Chinese academy of agricultural sciences, and the management measure is normal field management.

Experimental reagents and consumables:

enzyme and kit:

GXL DNA Polymerase high-fidelity enzyme, a fluorescence quantification kit, an RNA reverse transcription kit, a gel recovery kit and a PCR product purification kit are purchased from Takara bioengineering, Dalian, Co., Ltd;

the Ultra One Step Cloning Kit was purchased from Vazyme; the plasmid small quantity extraction kit is purchased from magenta company; restriction enzymes were purchased from NEB; the DNA Marker and the plant total RNA extraction kit are purchased from TIANGEN company.

Other drugs: agarose is a Spanish original product, peptone, yeast extract, chloroform, isoamylol, ethanol, isopropanol, sodium chloride and the like are domestic analytical purities, ampicillin and the like are purchased from Bao bioengineering Dalian Co., Ltd, and Escherichia coli competent cells are purchased from Beijing Tiangen Biochemical technology company.

Culture medium: LB liquid medium: 10g/L Tryptone (Tryptone), 5g/L Yeast extract (Yeast extract), and 10g/L sodium chloride (NaCl); LB solid medium: 10g/L of Tryptone (Tryptone), 5g/L of Yeast extract (Yeast extract), 10g/L of sodium chloride (NaCl) and 15g/L of agar powder, and the volume is fixed to 1L;

LB selective medium: before LB plate, adding antibiotic with corresponding concentration when the culture medium is sterilized under high pressure and cooled to 55 degree, shaking up and laying plate.

The main apparatus is as follows: PCR amplification apparatus (BIO-RAD), high speed centrifuge (Hettich MIKRO 200R), electrophoresis apparatus (BIO-RAD), gel imaging system (BIO-RAD), fluorescence quantitative PCR apparatus (ABI7500), electric heating constant temperature incubator (Shanghai Sensin), constant temperature culture oscillator (Shanghai Zhicheng), and artificial climate chamber.

Examples

1. Expression pattern analysis of cotton GhGPAT12/25 gene

Anthers at different periods were sampled, RNA extraction and reverse transcription of cDNA were performed, CDS sequence of GhGPAT12/25 was obtained from Cottongen, primers were designed, and fluorescent quantitative PCR was performed. FIG. 1 is a diagram showing the expression pattern of GhGPAT 12/25.

Cloning process:

(1) quickly freezing anther of upland cotton HM-1 material in different development stages in liquid nitrogen, grinding in liquid nitrogen, and storing in a refrigerator at-80 deg.C.

(2) Extracting total RNA of plants: the RNA extraction was performed using a TIANGEN RNA extraction kit.

(3) Synthesis of cDNA: and (2) carrying out reverse transcription on 500ng of RNA into cDNA by adopting a reverse transcription kit FSQ-201 of Toyobo, wherein a reverse transcription system is as follows:

RT reaction liquid is prepared according to the following components (the reaction liquid is prepared on ice):

TABLE 1 reverse transcription System

The reverse transcription reaction conditions were as follows:

15min at 37 ℃ (reverse transcription reaction),

5s at 98 ℃ (inactivation reaction of reverse transcriptase);

the reverse transcription product cDNA solution was diluted 6-fold as a template for PCR reaction.

(4) Fluorescent quantitative PCR

The primer sequence is as follows:

qGhGPAT12-F:5’-AAATCCTTGGAAAGGTGCCCCC-3’(SEQ ID No:7)；

qGhGPAT12-R:5’-ACTCAAATCCCAGTGCATCGGC-3’(SEQ ID No:8)；

qGhGPAT25-F:5’-GCAGGGCTGTTTTGCCAAAGTT-3’(SEQ ID No:9)；

qGhGPAT25-R:5’-GTGCAATTCAGTGCCTGCAACA-3’(SEQ ID No:10)。

and (3) PCR reaction system:

TABLE 2 fluorescent quantitative PCR reaction System

PCR reaction procedure:

TABLE 3 fluorescent quantitative PCR reaction procedure

Cloning of cotton GhGPAT12/25 gene

1.1 obtaining GhGPAT12/25 gene sequence from Cottongen, designing primer, amplifying CDS sequence of GhGPAT12/25 from anther cDNA of upland cotton HM-1 obtained by the above steps, wherein the open reading frame is 1626bp, codes 541 amino acids, the relative molecular weight of protein is 60.99/60.98kDa, and the isoelectric point is 9.12/9.29.

The amino acid sequence coded by GhGPAT12 is shown as the following SEQ ID No. 1:

MVFPVVFLKLADWVLYQLLANSCYRAARKVRNYGFFLRNQTPRSSSQQQAASLFPTASHCDVGNNIRSSQTLVCDIHGVLLRAETFFPYFMLVAFEAGGILRAFLLLLSCSFLWVLDYELKLRVMIFISFCGLRKKDIESVGRAVLPKFYLENLNLQAYEVWSKTSSRVVFTSIPRVMVEGFLKEYMAVDDVAGTELHTVGNRFTGLLSSSGLLVKHKALKAYFGDKKPDVGLGSSSLHDHHFISLCKEAYVVHKEDGRNNQSCLMPRDKYPKPLIFHDGRLAFLPTPFATLCMFLWLPFGIVLAIFRILVGICLPYRLAIFWGSLSGVQLTFQGCFPSSNLEQKKGVLYVCTHRTLLDPVFLSTALCKPLTAVTYSLSKMSEIIAPIKTVRLTRDRKQDGETMQKLLSEGDLVVCPEGTTCREPYLLRFSSLFAELADEIVPVAMNAHVSMFYGTTASGLKWLDPIFFLMNPRPSYHVQILGKVPPEFTCAGGRSSFEVANYIQRKLADALGFECTTFTRRDKYLMLAGNEGIVRENKRN；

the CDS sequence of the GhGPAT12 gene is shown as the following SEQ ID No. 2:

ATGGTTTTTCCTGTGGTATTTTTGAAGCTAGCTGACTGGGTCTTGTACCAGCTGCTGGCTAACTCATGTTATAGAGCCGCCAGGAAGGTGAGAAACTATGGTTTCTTTCTAAGGAACCAAACTCCTAGGTCGTCATCACAGCAGCAAGCTGCTTCTTTGTTCCCTACTGCTTCCCACTGTGATGTAGGCAACAATATTAGAAGCTCTCAAACACTGGTTTGTGATATTCATGGAGTCTTATTAAGAGCAGAAACCTTTTTCCCTTACTTCATGCTAGTTGCTTTTGAAGCTGGAGGCATTTTGAGAGCCTTTCTGTTGCTTTTATCGTGCTCCTTTTTGTGGGTTTTGGACTATGAGCTGAAATTGAGGGTAATGATTTTTATATCCTTCTGTGGGCTTAGGAAGAAGGACATTGAGAGCGTTGGCAGGGCTGTTTTGCCAAAGTTTTATCTTGAGAACCTTAATCTCCAAGCTTATGAAGTTTGGTCTAAAACAAGTTCAAGGGTTGTATTTACAAGTATACCTAGAGTTATGGTGGAAGGATTTCTCAAAGAATACATGGCCGTCGATGATGTTGCAGGCACTGAATTGCACACTGTTGGGAACCGATTCACAGGCTTGTTATCCAGCTCCGGTTTGCTGGTAAAGCACAAAGCTCTAAAGGCATACTTTGGTGATAAAAAGCCAGATGTTGGCCTCGGAAGTTCAAGCCTCCATGACCATCACTTTATCTCCCTTTGCAAGGAAGCCTATGTGGTGCACAAGGAAGATGGTAGAAACAATCAAAGTTGTTTGATGCCAAGGGACAAGTACCCAAAGCCACTTATATTTCATGATGGGAGACTAGCTTTCTTGCCAACACCTTTTGCAACACTCTGCATGTTCTTGTGGCTTCCATTTGGGATAGTTCTCGCTATATTTAGGATTCTGGTTGGTATCTGCCTGCCTTACAGGTTAGCTATTTTCTGGGGTTCTCTGAGTGGAGTACAGCTAACTTTCCAAGGCTGCTTTCCTTCATCCAATTTAGAACAGAAAAAAGGTGTTCTATATGTCTGTACCCATCGAACTCTTTTAGACCCAGTTTTCCTTAGCACAGCATTGTGCAAGCCTTTGACAGCCGTGACCTACAGCTTGAGCAAAATGTCTGAAATAATAGCTCCCATCAAGACAGTTAGGTTAACCAGGGACCGGAAGCAAGATGGAGAAACCATGCAAAAGTTGCTGAGTGAAGGTGATTTAGTGGTGTGTCCTGAGGGAACTACATGCAGAGAGCCTTATTTGTTAAGGTTTAGCTCATTGTTTGCTGAACTAGCGGATGAGATAGTTCCAGTAGCTATGAACGCTCATGTAAGTATGTTTTATGGAACAACTGCAAGTGGGTTGAAATGGTTGGATCCCATTTTCTTCCTGATGAACCCTAGACCTAGCTACCATGTTCAAATCCTTGGAAAGGTGCCCCCAGAGTTTACATGTGCCGGTGGCAGATCTAGCTTTGAAGTGGCAAATTATATTCAGAGAAAGCTGGCTGATGCACTGGGATTTGAGTGTACTACCTTTACTAGGAGAGACAAGTACTTGATGTTAGCAGGTAATGAAGGGATTGTCCGTGAAAATAAGAGAAATTGA；

the amino acid sequence coded by GhGPAT25 is shown as the following SEQ ID No. 3:

MVFPVVFLKLADWVLYQLLANSCYRAARKVRNYGFFLRNQIPRSSSQQQAASLFPSASHCDVGNNIRSSQTLVCDIHGVLLRAKTFFPYFMLLAFEAGGILRAFLLLLSCSFLWVLDYELKLRVMIFISFCGLRMKDIESVGRAVLPKFYLENLNLQAYEVWSKTSSRVVFTSIPRVMVEGFLKEYMAVDDVVGTELHTVGNRFTGLLSSSGLLVKHKALKAYFGDKKPDVGLGSSSLHDHHFISLCKEAYVVHKEDGRNNQSCLMPRDKYPKPLIFHDGRLAFLPTPFATLCMFLWLPFGIVLAIFRILVGICLPYRLAIFWGSLSGVQLTFQGCFPSSNSEQKKGVLYVCTHRTLLDPVFLSTALCKPLTAVTYSLSKMSEIIAPIKTVRLTRDRKQDGETMKKLLSEGDLVVCPEGTTCREPYLLRFSSLFAELADEIVPVAMSAHVSMFYGTTASGLKWLDPIFFLMNPRPSYHVQILGKVPPEFTCAGGRSSFEVANYIQRKLADALGFECTTFTRRDKYLMLAGNEGIVRENKRN；

the CDS sequence of the GhGPAT25 gene is shown as the following SEQ ID No. 4:

ATGGTTTTTCCTGTGGTATTTTTGAAGCTAGCGGACTGGGTCTTGTACCAGCTGCTGGCTAACTCATGTTATAGAGCCGCCAGGAAGGTGAGAAACTATGGGTTCTTTCTAAGGAACCAAATTCCTAGGTCGTCATCACAGCAGCAAGCTGCTTCTTTGTTCCCTAGTGCTTCCCACTGTGATGTAGGCAACAATATTAGAAGCTCTCAAACACTGGTTTGTGATATTCATGGAGTCTTATTAAGAGCAAAAACCTTTTTCCCTTACTTCATGCTACTTGCTTTTGAAGCTGGAGGCATTTTGAGAGCCTTTCTGTTGCTTTTATCGTGCTCCTTTTTGTGGGTTTTGGACTATGAGCTCAAATTGAGGGTAATGATTTTTATATCCTTCTGTGGGCTTAGGATGAAGGACATTGAGAGCGTTGGCAGGGCTGTTTTGCCAAAGTTTTATCTTGAGAACCTTAATCTCCAAGCTTATGAAGTTTGGTCTAAAACAAGTTCAAGGGTTGTGTTTACAAGTATACCTAGAGTTATGGTGGAAGGATTTCTCAAAGAATACATGGCCGTCGATGATGTTGTAGGAACTGAATTGCACACTGTTGGGAACCGATTCACAGGCTTGTTATCCAGCTCCGGTTTGCTGGTAAAGCACAAAGCTTTAAAGGCATACTTTGGTGATAAAAAGCCAGATGTTGGCCTCGGAAGTTCAAGCCTCCATGACCATCACTTTATCTCCCTTTGCAAGGAAGCCTATGTGGTGCACAAGGAAGATGGTAGAAACAATCAAAGTTGTTTGATGCCAAGGGACAAGTACCCGAAGCCACTTATATTTCATGATGGGAGACTAGCTTTCTTGCCAACACCATTTGCAACACTTTGCATGTTCTTGTGGCTTCCATTTGGGATAGTTCTCGCTATATTTAGGATTCTGGTTGGTATCTGCCTGCCTTACAGGTTAGCTATTTTCTGGGGTTCTCTGAGTGGAGTACAGCTAACTTTCCAAGGCTGCTTTCCTTCATCCAATTCAGAACAGAAAAAAGGTGTTCTATATGTCTGTACCCATCGAACTCTTTTAGACCCAGTTTTCCTTAGCACAGCATTGTGCAAGCCTTTGACAGCCGTGACCTACAGCTTGAGCAAAATGTCTGAAATAATAGCTCCCATCAAGACAGTTAGGTTAACCAGGGACCGGAAGCAAGATGGAGAAACCATGAAAAAGTTGCTGAGTGAAGGTGATTTAGTGGTGTGTCCTGAAGGAACTACATGCAGAGAGCCTTATTTGTTAAGGTTTAGTTCATTGTTTGCTGAACTAGCTGATGAGATAGTTCCAGTAGCCATGAGCGCTCATGTAAGTATGTTTTATGGAACAACTGCAAGTGGGTTGAAATGGTTGGATCCCATTTTCTTCCTGATGAACCCTAGACCTAGCTACCATGTTCAAATCCTTGGAAAGGTGCCCCCAGAGTTTACATGTGCCGGGGGCAGATCTAGCTTTGAAGTGGCAAATTATATTCAGAGAAAGTTGGCCGATGCACTGGGATTTGAGTGTACTACCTTTACTAGGAGAGACAAGTACTTGATGTTAGCAGGTAATGAAGGGATTGTCCGTGAAAATAAGAGAAATTGA。

PCR amplification of target genes

The following system was prepared on ice, and the desired gene GhGPAT12/25 was amplified using cDNA of HM-1 as a template. According to TaKaRa

GXL DNA Polymerase highThe following PCR reaction system is adopted in the fidelity enzyme specification:

TABLE 4 PCR amplification reaction System

The PCR amplification procedure was: 3min at 98 ℃; 10s at 98 ℃; 15s at 56 ℃; 1min at 68 ℃ for 35 cycles; 10min at 68 ℃.

The primer sequence is as follows:

GhGPAT12/25-F:5′-ATGGTTTTTCCTGTGGTAT-3′(SEQ ID No:11)；

GhGPAT12/25-R:5′-TCAATTTCTCTTATTTTCA-3′(SEQ ID No:12)。

after the reaction was completed, the reaction mixture was stored at 4 ℃.

(5) And (3) cutting and recovering the target fragment by using a gel recovery kit.

(6) The product recovered from the above gum is

The Ultra One Step Cloning Kit is used for constructing a connection T vector and transforming Escherichia coli.

(7) After picking single clone from the resistant LB culture medium overnight at 37 ℃, the culture was carried out with shaking at 37 ℃.

(8) And (3) carrying out PCR verification on bacterial liquid, selecting a positive clone sample, sending the sample to Jinzhi biotechnology limited for sequencing, and adding a certain amount of glycerol into the bacterial liquid with correct sequencing to ensure that the final concentration of the glycerol is about 20 percent and storing the glycerol at-70 ℃.

GhGPAT12/25-CRISPR vector construction

2.1 target sequence determination and primer design

Designing 2 CRISPR target sites according to the provided mRNA sequence and corresponding genome sequence information, and designing a PCR amplification primer according to the target sites; the corresponding primers were ligated to in-fusion linkers and used for subsequent ligation experiments after synthesis.

TABLE 5 target site sequences

TABLE 6PCR amplification primers

2.2 amplification of the fragment of interest

The overlap extension PCR is adopted to amplify the fragment containing the target site, and the reaction system is as follows:

PCR system

Table 7 target fragment amplification reaction System

The PCR procedure was:

2.3 vector construction

The vector pRGEB32-GhU6.9-NPT II is digested by BsaI, and the digestion system is as follows:

TABLE 8 enzyme digestion System

After amplification, the desired fragment was ligated to the cleaved pRGEB32-GhU6.9-NPT II vector

TABLE 9 In-fusion ligation reaction System

Water bath at 37 deg.C for 30min, and standing on ice for 5min for storage at-20 deg.C.

2.4 transformation of Escherichia coli by electric shock

The constructed CRISPR vector was electroporated into escherichia coli TOP10 and positive clones were screened by colony PCR. The detection primer is U6-7 s: TGTGCCACTCCAAAGACATCAG (SEQ ID No:17), GhGPAT12/25-inf-T2 as: TTCTAGCTCTAAAACTGCCAACGCTCTCAATGTCC (SEQ ID No: 16). The positive clone detection method is as follows:

and (3) PCR system:

TABLE 10 Positive clone detection PCR System

PCR procedure:

1 positive clone was picked for sequencing by the CRISPR vector.

3. Genetic transformation of cotton stem segments using Agrobacterium mediation

A. Transforming the positive clone into an agrobacterium-sensitive strain GV3101, carrying out amplification culture on agrobacterium, centrifuging, discarding supernatant, adding invasive stain solution (MGL and AS), vibrating to suspend the bacterium solution, and activating at least 30min at a shaking table at 28 ℃ and 200 rpm/min;

B. sterilizing acceptor cotton (HM-1) seeds with mercuric chloride, cleaning with sterile water, placing into sterile seedling culture medium, and culturing at 30 deg.C for 6 d;

C. cutting the hypocotyl of the acceptor seedling into small stem sections, infecting the small stem sections with activated agrobacterium and drying the small stem sections;

D. laying the hypocotyl in a co-culture medium containing filter paper, and performing dark culture at 20 ℃ for 1-2 d;

E. transferring the hypocotyl into a 2,4-D culture medium, placing the hypocotyl into a light culture chamber, and carrying out subculture for about 20-30 days;

F. growing the callus into rice-grain-shaped particles, transferring the rice-grain-shaped particles into a differentiation culture medium, and further differentiating into embryoids;

G. subculturing the differentiated plantlets into a rooting culture medium until the plantlets grow into plantlets with good and healthy roots;

H. transferring the seedlings into water, hardening the seedlings, and planting the seedlings in a greenhouse after about one week.

4. Detection of Gene editing Condition in transgenic Cotton plants

4.1 PCR detection of transgenic plants

Obtaining 15 transgenic single plants in total, cutting regenerated plant leaves, extracting DNA by a CTAB method, and performing PCR detection by using an nptII specific primer.

TABLE 11 transgenic plant detection primers

And (3) PCR system:

TABLE 12 transgenic plant detection reaction system

PCR reaction procedure:

FIG. 2 shows the result chart of PCR detection of cotton transgenic plants, and 13 positive individuals are found in the detection result, and are numbered as 6-1, 6-2, 6-6, 6-7, 6-9, 6-11, 6-12, 6-15, 6-23, 6-25, 6-26, 6-28 and 6-29.

4.3 second-generation sequencing detection of transgenic plant editing conditions

Designing primers based on the reference sequence to amplify the sequence between the two target points, and amplifying with the wild type sequence. The amplified primer sequences were as follows:

TABLE 13 amplification primers for products to be sequenced

Detection system and program

The amplification system was as follows: primaceae T3 Mix 27 μ l, Primer F (10 μ M)0.5 μ l, Primer R (10 μ M)0.5 μ l, DNA template 1.5 μ l, and water to 30 μ l. The reaction program is 98 ℃ for 2 min; 10s at 98 ℃, 10s at 56 ℃, 10s at 72 ℃ and 30 cycles; 5min at 72 ℃; at25 ℃ for 2 min.

The primers in Table 1 were used to perform PCR amplification and library construction on the transgenic samples.

Mixing the PCR amplified library samples, digging and recovering gel, determining the concentration of the samples, performing high-throughput sequencing on the samples, analyzing sequencing data by taking the provided sequence as a reference genome sequence, and obtaining the gene sequence type of the target site section in each sample and detailed sequence information thereof.

The results showed that the GhGPAT12/25 gene of 6-2, 6-9, 6-12, 6-15, 6-23, 6-25, 6-26, 6-28, 6-29 transgenic plants was completely edited, 6-1 contained the unedited GhGPAT12 wild-type sequence, 6-7 contained the unedited GhGPAT25 wild-type sequence, and 6-6, 6-11 contained the unedited GhGPAT12/25 wild-type sequence.

5. Phenotypic identification of transgenic plants

Phenotypic observations of transgenic individuals of different editing types are shown in FIG. 3, where A-D are transgenic T₀Generation individuals, wherein A is fertile 6-1 individuals, B is sterile 6-15 individuals, C is fertile 6-11 individuals, and D is fertile 6-7 individuals; E-J is transgenic hybrid T₂The generation individual plant, E, G, I is the WT plant, flower organ and pollen stain, F is the sterile plant, flower organ and pollen stain.

The fully edited individual to GhGPAT12/25 was found to be completely sterile, in particular represented by shortened filaments and shriveled anthers, and no mature pollen grains were found in the anthers by starch-potassium iodide staining. While the single strain containing the wild GhGPAT12 or/and GhGPAT25 sequence shows normalAnd (4) fertility. Alternatively, 6-15 transgenic individuals were crossed with WT, followed by F₂And carrying out phenotype observation on the generation plants. As a result, it was found that F₂The individual plants in the generation are separated from each other by the fertility phenotype, the phenotype of the sterile individual plant is separated from T₀Generations are similar.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

<110> Cotton research institute of Chinese academy of agricultural sciences

<130> PA21000811

<160> 21

<170> PatentIn version 3.3

<210> 1

<211> 541

<212> PRT

<213> amino acid GhGPAT12

<400> 1

Met Val Phe Pro Val Val Phe Leu Lys Leu Ala Asp Trp Val Leu Tyr

1 5 10 15

Gln Leu Leu Ala Asn Ser Cys Tyr Arg Ala Ala Arg Lys Val Arg Asn

20 25 30

Tyr Gly Phe Phe Leu Arg Asn Gln Thr Pro Arg Ser Ser Ser Gln Gln

35 40 45

Gln Ala Ala Ser Leu Phe Pro Thr Ala Ser His Cys Asp Val Gly Asn

50 55 60

Asn Ile Arg Ser Ser Gln Thr Leu Val Cys Asp Ile His Gly Val Leu

65 70 75 80

Leu Arg Ala Glu Thr Phe Phe Pro Tyr Phe Met Leu Val Ala Phe Glu

85 90 95

Ala Gly Gly Ile Leu Arg Ala Phe Leu Leu Leu Leu Ser Cys Ser Phe

100 105 110

Leu Trp Val Leu Asp Tyr Glu Leu Lys Leu Arg Val Met Ile Phe Ile

115 120 125

Ser Phe Cys Gly Leu Arg Lys Lys Asp Ile Glu Ser Val Gly Arg Ala

130 135 140

Val Leu Pro Lys Phe Tyr Leu Glu Asn Leu Asn Leu Gln Ala Tyr Glu

145 150 155 160

Val Trp Ser Lys Thr Ser Ser Arg Val Val Phe Thr Ser Ile Pro Arg

165 170 175

Val Met Val Glu Gly Phe Leu Lys Glu Tyr Met Ala Val Asp Asp Val

180 185 190

Ala Gly Thr Glu Leu His Thr Val Gly Asn Arg Phe Thr Gly Leu Leu

195 200 205

Ser Ser Ser Gly Leu Leu Val Lys His Lys Ala Leu Lys Ala Tyr Phe

210 215 220

Gly Asp Lys Lys Pro Asp Val Gly Leu Gly Ser Ser Ser Leu His Asp

225 230 235 240

His His Phe Ile Ser Leu Cys Lys Glu Ala Tyr Val Val His Lys Glu

245 250 255

Asp Gly Arg Asn Asn Gln Ser Cys Leu Met Pro Arg Asp Lys Tyr Pro

260 265 270

Lys Pro Leu Ile Phe His Asp Gly Arg Leu Ala Phe Leu Pro Thr Pro

275 280 285

Phe Ala Thr Leu Cys Met Phe Leu Trp Leu Pro Phe Gly Ile Val Leu

290 295 300

Ala Ile Phe Arg Ile Leu Val Gly Ile Cys Leu Pro Tyr Arg Leu Ala

305 310 315 320

Ile Phe Trp Gly Ser Leu Ser Gly Val Gln Leu Thr Phe Gln Gly Cys

325 330 335

Phe Pro Ser Ser Asn Leu Glu Gln Lys Lys Gly Val Leu Tyr Val Cys

340 345 350

Thr His Arg Thr Leu Leu Asp Pro Val Phe Leu Ser Thr Ala Leu Cys

355 360 365

Lys Pro Leu Thr Ala Val Thr Tyr Ser Leu Ser Lys Met Ser Glu Ile

370 375 380

Ile Ala Pro Ile Lys Thr Val Arg Leu Thr Arg Asp Arg Lys Gln Asp

385 390 395 400

Gly Glu Thr Met Gln Lys Leu Leu Ser Glu Gly Asp Leu Val Val Cys

405 410 415

Pro Glu Gly Thr Thr Cys Arg Glu Pro Tyr Leu Leu Arg Phe Ser Ser

420 425 430

Leu Phe Ala Glu Leu Ala Asp Glu Ile Val Pro Val Ala Met Asn Ala

435 440 445

His Val Ser Met Phe Tyr Gly Thr Thr Ala Ser Gly Leu Lys Trp Leu

450 455 460

Asp Pro Ile Phe Phe Leu Met Asn Pro Arg Pro Ser Tyr His Val Gln

465 470 475 480

Ile Leu Gly Lys Val Pro Pro Glu Phe Thr Cys Ala Gly Gly Arg Ser

485 490 495

Ser Phe Glu Val Ala Asn Tyr Ile Gln Arg Lys Leu Ala Asp Ala Leu

500 505 510

Gly Phe Glu Cys Thr Thr Phe Thr Arg Arg Asp Lys Tyr Leu Met Leu

515 520 525

Ala Gly Asn Glu Gly Ile Val Arg Glu Asn Lys Arg Asn

530 535 540

<210> 2

<211> 1626

<212> DNA

<213> GhGPAT12 CDS

<400> 2

atggtttttc ctgtggtatt tttgaagcta gctgactggg tcttgtacca gctgctggct 60

aactcatgtt atagagccgc caggaaggtg agaaactatg gtttctttct aaggaaccaa 120

actcctaggt cgtcatcaca gcagcaagct gcttctttgt tccctactgc ttcccactgt 180

gatgtaggca acaatattag aagctctcaa acactggttt gtgatattca tggagtctta 240

ttaagagcag aaaccttttt cccttacttc atgctagttg cttttgaagc tggaggcatt 300

ttgagagcct ttctgttgct tttatcgtgc tcctttttgt gggttttgga ctatgagctg 360

aaattgaggg taatgatttt tatatccttc tgtgggctta ggaagaagga cattgagagc 420

gttggcaggg ctgttttgcc aaagttttat cttgagaacc ttaatctcca agcttatgaa 480

gtttggtcta aaacaagttc aagggttgta tttacaagta tacctagagt tatggtggaa 540

ggatttctca aagaatacat ggccgtcgat gatgttgcag gcactgaatt gcacactgtt 600

gggaaccgat tcacaggctt gttatccagc tccggtttgc tggtaaagca caaagctcta 660

aaggcatact ttggtgataa aaagccagat gttggcctcg gaagttcaag cctccatgac 720

catcacttta tctccctttg caaggaagcc tatgtggtgc acaaggaaga tggtagaaac 780

aatcaaagtt gtttgatgcc aagggacaag tacccaaagc cacttatatt tcatgatggg 840

agactagctt tcttgccaac accttttgca acactctgca tgttcttgtg gcttccattt 900

gggatagttc tcgctatatt taggattctg gttggtatct gcctgcctta caggttagct 960

attttctggg gttctctgag tggagtacag ctaactttcc aaggctgctt tccttcatcc 1020

aatttagaac agaaaaaagg tgttctatat gtctgtaccc atcgaactct tttagaccca 1080

gttttcctta gcacagcatt gtgcaagcct ttgacagccg tgacctacag cttgagcaaa 1140

atgtctgaaa taatagctcc catcaagaca gttaggttaa ccagggaccg gaagcaagat 1200

ggagaaacca tgcaaaagtt gctgagtgaa ggtgatttag tggtgtgtcc tgagggaact 1260

acatgcagag agccttattt gttaaggttt agctcattgt ttgctgaact agcggatgag 1320

atagttccag tagctatgaa cgctcatgta agtatgtttt atggaacaac tgcaagtggg 1380

ttgaaatggt tggatcccat tttcttcctg atgaacccta gacctagcta ccatgttcaa 1440

atccttggaa aggtgccccc agagtttaca tgtgccggtg gcagatctag ctttgaagtg 1500

gcaaattata ttcagagaaa gctggctgat gcactgggat ttgagtgtac tacctttact 1560

aggagagaca agtacttgat gttagcaggt aatgaaggga ttgtccgtga aaataagaga 1620

aattga 1626

<210> 3

<211> 541

<212> PRT

<213> amino acid GhGPAT25

<400> 3

Met Val Phe Pro Val Val Phe Leu Lys Leu Ala Asp Trp Val Leu Tyr

1 5 10 15

Gln Leu Leu Ala Asn Ser Cys Tyr Arg Ala Ala Arg Lys Val Arg Asn

20 25 30

Tyr Gly Phe Phe Leu Arg Asn Gln Ile Pro Arg Ser Ser Ser Gln Gln

35 40 45

Gln Ala Ala Ser Leu Phe Pro Ser Ala Ser His Cys Asp Val Gly Asn

50 55 60

Asn Ile Arg Ser Ser Gln Thr Leu Val Cys Asp Ile His Gly Val Leu

65 70 75 80

Leu Arg Ala Lys Thr Phe Phe Pro Tyr Phe Met Leu Leu Ala Phe Glu

85 90 95

Ala Gly Gly Ile Leu Arg Ala Phe Leu Leu Leu Leu Ser Cys Ser Phe

100 105 110

Leu Trp Val Leu Asp Tyr Glu Leu Lys Leu Arg Val Met Ile Phe Ile

115 120 125

Ser Phe Cys Gly Leu Arg Met Lys Asp Ile Glu Ser Val Gly Arg Ala

130 135 140

Val Leu Pro Lys Phe Tyr Leu Glu Asn Leu Asn Leu Gln Ala Tyr Glu

145 150 155 160

Val Trp Ser Lys Thr Ser Ser Arg Val Val Phe Thr Ser Ile Pro Arg

165 170 175

Val Met Val Glu Gly Phe Leu Lys Glu Tyr Met Ala Val Asp Asp Val

180 185 190

Val Gly Thr Glu Leu His Thr Val Gly Asn Arg Phe Thr Gly Leu Leu

195 200 205

Ser Ser Ser Gly Leu Leu Val Lys His Lys Ala Leu Lys Ala Tyr Phe

210 215 220

Gly Asp Lys Lys Pro Asp Val Gly Leu Gly Ser Ser Ser Leu His Asp

225 230 235 240

His His Phe Ile Ser Leu Cys Lys Glu Ala Tyr Val Val His Lys Glu

245 250 255

Asp Gly Arg Asn Asn Gln Ser Cys Leu Met Pro Arg Asp Lys Tyr Pro

260 265 270

Lys Pro Leu Ile Phe His Asp Gly Arg Leu Ala Phe Leu Pro Thr Pro

275 280 285

Phe Ala Thr Leu Cys Met Phe Leu Trp Leu Pro Phe Gly Ile Val Leu

290 295 300

Ala Ile Phe Arg Ile Leu Val Gly Ile Cys Leu Pro Tyr Arg Leu Ala

305 310 315 320

Ile Phe Trp Gly Ser Leu Ser Gly Val Gln Leu Thr Phe Gln Gly Cys

325 330 335

Phe Pro Ser Ser Asn Ser Glu Gln Lys Lys Gly Val Leu Tyr Val Cys

340 345 350

Thr His Arg Thr Leu Leu Asp Pro Val Phe Leu Ser Thr Ala Leu Cys

355 360 365

Lys Pro Leu Thr Ala Val Thr Tyr Ser Leu Ser Lys Met Ser Glu Ile

370 375 380

Ile Ala Pro Ile Lys Thr Val Arg Leu Thr Arg Asp Arg Lys Gln Asp

385 390 395 400

Gly Glu Thr Met Lys Lys Leu Leu Ser Glu Gly Asp Leu Val Val Cys

405 410 415

Pro Glu Gly Thr Thr Cys Arg Glu Pro Tyr Leu Leu Arg Phe Ser Ser

420 425 430

Leu Phe Ala Glu Leu Ala Asp Glu Ile Val Pro Val Ala Met Ser Ala

435 440 445

His Val Ser Met Phe Tyr Gly Thr Thr Ala Ser Gly Leu Lys Trp Leu

450 455 460

Asp Pro Ile Phe Phe Leu Met Asn Pro Arg Pro Ser Tyr His Val Gln

465 470 475 480

Ile Leu Gly Lys Val Pro Pro Glu Phe Thr Cys Ala Gly Gly Arg Ser

485 490 495

Ser Phe Glu Val Ala Asn Tyr Ile Gln Arg Lys Leu Ala Asp Ala Leu

500 505 510

Gly Phe Glu Cys Thr Thr Phe Thr Arg Arg Asp Lys Tyr Leu Met Leu

515 520 525

Ala Gly Asn Glu Gly Ile Val Arg Glu Asn Lys Arg Asn

530 535 540

<210> 4

<211> 1626

<212> DNA

<213> GhGPAT25 CDS

<400> 4

atggtttttc ctgtggtatt tttgaagcta gcggactggg tcttgtacca gctgctggct 60

aactcatgtt atagagccgc caggaaggtg agaaactatg ggttctttct aaggaaccaa 120

attcctaggt cgtcatcaca gcagcaagct gcttctttgt tccctagtgc ttcccactgt 180

gatgtaggca acaatattag aagctctcaa acactggttt gtgatattca tggagtctta 240

ttaagagcaa aaaccttttt cccttacttc atgctacttg cttttgaagc tggaggcatt 300

ttgagagcct ttctgttgct tttatcgtgc tcctttttgt gggttttgga ctatgagctc 360

aaattgaggg taatgatttt tatatccttc tgtgggctta ggatgaagga cattgagagc 420

gttggcaggg ctgttttgcc aaagttttat cttgagaacc ttaatctcca agcttatgaa 480

gtttggtcta aaacaagttc aagggttgtg tttacaagta tacctagagt tatggtggaa 540

ggatttctca aagaatacat ggccgtcgat gatgttgtag gaactgaatt gcacactgtt 600

gggaaccgat tcacaggctt gttatccagc tccggtttgc tggtaaagca caaagcttta 660

aaggcatact ttggtgataa aaagccagat gttggcctcg gaagttcaag cctccatgac 720

catcacttta tctccctttg caaggaagcc tatgtggtgc acaaggaaga tggtagaaac 780

aatcaaagtt gtttgatgcc aagggacaag tacccgaagc cacttatatt tcatgatggg 840

agactagctt tcttgccaac accatttgca acactttgca tgttcttgtg gcttccattt 900

gggatagttc tcgctatatt taggattctg gttggtatct gcctgcctta caggttagct 960

attttctggg gttctctgag tggagtacag ctaactttcc aaggctgctt tccttcatcc 1020

aattcagaac agaaaaaagg tgttctatat gtctgtaccc atcgaactct tttagaccca 1080

gttttcctta gcacagcatt gtgcaagcct ttgacagccg tgacctacag cttgagcaaa 1140

atgtctgaaa taatagctcc catcaagaca gttaggttaa ccagggaccg gaagcaagat 1200

ggagaaacca tgaaaaagtt gctgagtgaa ggtgatttag tggtgtgtcc tgaaggaact 1260

acatgcagag agccttattt gttaaggttt agttcattgt ttgctgaact agctgatgag 1320

atagttccag tagccatgag cgctcatgta agtatgtttt atggaacaac tgcaagtggg 1380

ttgaaatggt tggatcccat tttcttcctg atgaacccta gacctagcta ccatgttcaa 1440

atccttggaa aggtgccccc agagtttaca tgtgccgggg gcagatctag ctttgaagtg 1500

gcaaattata ttcagagaaa gttggccgat gcactgggat ttgagtgtac tacctttact 1560

aggagagaca agtacttgat gttagcaggt aatgaaggga ttgtccgtga aaataagaga 1620

aattga 1626

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence

<400> 5

actcatgtta tagagccgcc 20

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence

<400> 6

ggacattgag agcgttggca 20

<210> 7

<211> 22

<212> DNA

<213> Artificial sequence

<400> 7

aaatccttgg aaaggtgccc cc 22

<210> 8

<211> 22

<212> DNA

<213> Artificial sequence

<400> 8

actcaaatcc cagtgcatcg gc 22

<210> 9

<211> 22

<212> DNA

<213> Artificial sequence

<400> 9

gcagggctgt tttgccaaag tt 22

<210> 10

<211> 22

<212> DNA

<213> Artificial sequence

<400> 10

gtgcaattca gtgcctgcaa ca 22

<210> 11

<211> 19

<212> DNA

<213> Artificial sequence

<400> 11

atggtttttc ctgtggtat 19

<210> 12

<211> 19

<212> DNA

<213> Artificial sequence

<400> 12

tcaatttctc ttattttca 19

<210> 13

<211> 36

<212> DNA

<213> Artificial sequence

<400> 13

ggcggctcta taacatgagt tgcaccagcc gggaat 36

<210> 14

<211> 38

<212> DNA

<213> Artificial sequence

<400> 14

actcatgtta tagagccgcc gttttagagc tagaaata 38

<210> 15

<211> 36

<212> DNA

<213> Artificial sequence

<400> 15

tgccaacgct ctcaatgtcc tgcaccagcc gggaat 36

<210> 16

<211> 35

<212> DNA

<213> Artificial sequence

<400> 16

ttctagctct aaaactgcca acgctctcaa tgtcc 35

<210> 17

<211> 22

<212> DNA

<213> Artificial sequence

<400> 17

tgtgccactc caaagacatc ag 22

<210> 18

<211> 21

<212> DNA

<213> Artificial sequence

<400> 18

actgggcaca acagacaatc g 21

<210> 19

<211> 23

<212> DNA

<213> Artificial sequence

<400> 19

gcatcagcca tgatggatac ttt 23

<210> 20

<211> 24

<212> DNA

<213> Artificial sequence

<400> 20

tggtttttcc tgtggtattt ttga 24

<210> 21

<211> 24

<212> DNA

<213> Artificial sequence

<400> 21

aaatccttcc accataactc tagg 24

Claims

The application of GhGPAT12 protein and GhGPAT25 protein in regulation and control of cotton male reproductive development is characterized in that the amino acid sequence of the GhGPAT12 protein is shown as SEQ ID No. 1; the amino acid sequence of the GhGPAT25 protein is shown in SEQ ID No. 3.
The application of GhGPAT12 protein and GhGPAT25 protein in breeding male sterile transgenic cotton is characterized in that the amino acid sequence of the GhGPAT12 protein is shown as SEQ ID No. 1; the amino acid sequence of the GhGPAT25 protein is shown in SEQ ID No. 3.
The application of the coding genes of GhGPAT12 protein and GhGPAT25 protein or the biological material containing the coding genes in regulation of cotton male reproductive development, pollen development or cultivation of male sterile transgenic cotton is characterized in that the CDS sequence of the coding gene of the GhGPAT12 protein is shown as SEQ ID No. 2; the CDS sequence of the coding gene of the GhGPAT25 protein is shown as SEQ ID No. 4.
4. Use according to claim 3, wherein the biological material comprises an expression cassette, an expression vector or a host cell.
5. The use of any one of claims 1 to 4, wherein the use is by inactivating the genes encoding the GhGPAT12 protein and the GhGPAT25 protein, or by reducing or inhibiting the expression of the genes encoding the GhGPAT12 protein and the GhGPAT25 protein.
6. The application of claim 5, wherein the application comprises inactivating genes encoding GhGPAT12 protein and GhGPAT25 protein, or reducing or inhibiting expression of the genes encoding GhGPAT12 protein and GhGPAT25 protein by gene knockout, gene knockdown or gene editing, so that the mutant cotton has male sterility.
7. The application of claim 6, wherein the CRISPR/Cas9 system construction vector is adopted for gene editing of coding genes of GhGPAT12 protein and GhGPAT25 protein.
8. The use according to claim 7, wherein the target site sequence of the designed CRISPR/Cas9 vector is shown as SEQ ID No. 5 and SEQ ID No. 6.
9. A method for breeding male sterile line transgenic cotton is characterized in that the method comprises the step of obtaining a male sterile line transgenic plant by inactivating coding genes of GhGPAT12 protein and GhGPAT25 protein or reducing or inhibiting the expression of the coding genes of GhGPAT12 protein and GhGPAT25 protein.
10. The method of claim 9, wherein the cotton comprises gossypium barbadense and gossypium hirsutum, preferably gossypium hirsutum.