CN117603991A - Gene OsGSL2 and application of mutant OsGSL2-3 thereof in rice male fertility regulation - Google Patents

Abstract

The invention relates to the field of plant biotechnology breeding, and provides application of a gene OsGSL2 and a mutant OsGSL2-3 thereof in rice male fertility regulation. The male fertility control sterile gene OsGSL2 has a nucleotide sequence shown as SEQ ID NO.1, and the encoded protein has an amino acid sequence shown as SEQ ID NO. 2. The invention mutates the OsGSL2 gene in a fixed point in wild rice, and the mutant OsGSL2-3 has the advantages that although anther can be normally formed, pollen is completely aborted and no pollen particle is formed, so that the regulation and control function of the OsGSL2 gene on male reproductive development of rice is discovered for the first time, and the invention has important significance on researching the male reproductive development of rice.

Description

Gene OsGSL2 and application of mutant OsGSL2-3 thereof in rice male fertility regulation

Technical Field

The invention relates to the field of plant biotechnology breeding, in particular to application of a gene OsGSL2 and a mutant OsGSL2-3 thereof in rice male fertility regulation and control.

Background

Rice is one of the most important food crops in the world. With the growth of population and the improvement of life quality, the annual yield of the rice in 2050 is expected to be improved by 1-2 times so as to meet the requirement of human development. Hybrid rice is a child generation obtained after parent-parent hybridization, the yield of the hybrid rice is often improved by more than 15% compared with that of a conventional rice parent, and the resistance and the adaptability are far superior to those of the parent. Therefore, application and popularization of hybrid rice are an important way to increase rice yield.

The male sterile line is a key node of hybrid rice seed production technology. Male sterile line refers to a plant line in which male gametes are dysplastic and lose fertility and female gametes are normal. It can only be used as female parent to accept pollen of male parent, and selfing can not be firm. The male sterile line applied to the production of the hybrid rice at present has two types of nuclear-cytoplasmic interaction type and photo-thermo-sensitive type. The sterile gene of the nuclear-cytoplasmic interactive male sterile line is in cytoplasm and the nucleus has no fertility restoration gene. When the restoring line with fertility restoring gene in cell nucleus is hybridized with its matched group, it can produce the first generation hybrid seed, when the maintaining line without fertility restoring gene in cell nucleus and without sterile gene in cell cytoplasm is hybridized with it, it can reproduce the sterile line seed. The hybrid rice seed production technique is often called a three-line method because of the need of the matching of sterile line, maintainer line and restorer line. Several genes controlling nuclear cytoplasmic interactive sterility and corresponding fertility restoration have been cloned (Chen and Liu,2014,Male sterility and fertility restoration in crops,Annu Rev Plant Biol,65:579-606). The nuclear-cytoplasm interactive sterile line is the first sterile line applied on a large scale in hybrid rice seed production, and lays a material foundation for the establishment and development of hybrid rice industry. However, since the assembly of the cytoplasmic interactive sterile line is limited by the restorer genotype, only about 5% of the germplasm resources can be utilized. While cytoplasmic sterile genes have the potential to cause poor rice quality and the prevalence of specific diseases and pests.

The photo-thermo-sensitive male sterile line is a sterile line with fertility regulated by photo-thermo environment. The sterile line is kept sterile under a certain light temperature condition, and can be used for combined hybridization. When the conditions change, the sterile line restores fertility and can be used for sterile line propagation. Because the photo-thermo-sensitive male sterile line realizes the combination of the sterile line and the maintainer line, only the male parent is matched with the male parent to produce the first filial generation hybrid, so the corresponding breeding technology is often called a two-line method. Genes regulating photo-thermo-sensitive male sterility in nuclei, genes that have been cloned so far include PMS3, TMS5, CSA and TMS10 (Chen and Liu,2014,Male sterility and fertility restoration in crops,Annu Rev Plant Biol,65:579-606;Zhou H,et al,2014,RNase ZS1 processes UbL40 mRNAs and controls thermosensitive genic male sterility in rice,Nature Communications,5:4884-4892). Compared with the nuclear-cytoplasmic interactive sterile line, the photo-thermo-sensitive sterile line has simple propagation procedure and more free matching due to the wide existence of the restoring gene. The large-scale application of photo-thermo-sensitive sterile line greatly consolidates and promotes the development of hybrid rice industry. However, the fertility of the sterile line is affected by the light and temperature environment, so that the risk of seed production is high, and the seed production region is limited.

In order to overcome the key defects existing in the current hybrid rice seed production technology, the creation and utilization of a new type of sterile line is an important break. The nuclear male sterility is generated by nuclear gene mutation, and has dominant mutation, recessive mutation, sporophyte gene mutation and gametophyte gene mutation. Dominant mutations and gametophytic gene mutations can only be inherited through female gametes, recessive mutations can be inherited through both female gametes and male gametes, and follow Mendelian's law. The invention provides a plant fertility regulating gene and a recessive nuclear sterile type male sterile line based on the gene mutation. The sterile line has stable fertility, is only regulated and controlled by a single gene of nuclear coding, and is not influenced by light temperature environment. The fertility restorer gene of the sterile line is widely existed in rice germplasm resources, and can restore fertility by transferring wild type genes. The gene and the sterile line generated by the mutation of the gene provide elements for developing novel hybrid seed production technology of rice, and lay a foundation for solving the problems existing in the prior art.

Callose synthases are polysaccharides consisting mainly of beta-1, 3-glucan, providing structural support and protection to plant cells. The deposition of callose occurs in a wide range of biological processes, from the development of plant environmental stress reactions. Callose synthases accumulate in the cell wall of dividing cells prior to cell wall formation, and callose accumulates internally on the surface of the wall adjacent to the plasma membrane during plant stress. Furthermore, callose is present in phloem sieve pores, which is the primary channel that mediates the long-range movement of signaling molecules and nutrients. Callose is also thought to be located in the neck of plasmodesmata and regulates inter-cell communication.

Compared with the mode plant arabidopsis thaliana and the mode crop rice, the rice has relatively fewer cloned and identified common nuclear male sterile genes and created male sterile materials. The CRISPR/Cas9 (Clustered, regularly Interspaced, short Palindromic Repeatsassociated Endonuclease 9) gene editing technology has the characteristics of low cost, simple operation, high mutation induction rate and the like, and is more and more widely applied to the aspects of plant gene function research, crop genetic improvement, breeding and the like, so that the application prospect is very broad.

The invention utilizes CRISPR/Cas9 technology to excavate and identify rice male sterile candidate genes and create male sterile materials, can rapidly enrich rice common nuclear male sterile genes and sterile material resources, thereby promoting rice sterile breeding and seed production popularization and application, and finally can effectively solve the bottleneck problem of short stable rice sterile lines and breakthrough large varieties in the rice seed industry for a long time.

Disclosure of Invention

The invention aims to provide an application of a gene OsGSL2 and a mutant OsGSL2-3 thereof in rice male fertility regulation.

In order to achieve the purpose of the invention, in a first aspect, the invention provides an application of a gene OsGSL2 in rice male fertility regulation, wherein the gene OsGSL2 is a gene encoding the following protein (a) or (b):

(a) A protein consisting of the amino acid sequence shown in SEQ ID NO. 2; or (b)

(b) And (b) a protein which is derived from (a) and has equivalent functions and is obtained by substituting, deleting or adding one or more amino acids in the sequence shown in SEQ ID NO. 2.

The nucleotide sequence of the gene OsGSL2 is shown as SEQ ID NO.1 (LOC_Os01g 48200, see rice genome database http:// rice. Uga. Edu /).

Further, by modifying the gene OsGSL2, the function of the gene is deleted or weakened, so that the rice obtains the male sterile character.

In a second aspect, the present invention provides a method of creating a male sterile line of rice, the method comprising: inhibiting the expression and/or activity of gene OsGSL2 in rice plants.

Means for inhibiting the expression and/or activity of gene OsGSL2 include, but are not limited to, gene editing techniques, or introducing into rice a molecule having the expression and/or activity of gene OsGSL2.

The molecule with the gene OsGSL2 expression and/or activity is selected from at least one of shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA/DNA, low molecular compounds, peptides, antibodies and the like.

Such gene editing techniques include, but are not limited to, CRISPR, TALEN, and ZFN.

Preferably, CRISPR/Cas9 gene editing is utilized to inhibit the expression and/or activity of OsGSL2 genes.

In a third aspect, the invention provides a CRISPR/Cas9 system-based sgRNA of a targeting gene OsGSL2, wherein the sgRNA targets exon 1 of the gene OsGSL2, and the nucleotide sequence of an action site is 5'-GAGCAGAGACGTTTATAGACTGG-3' (SEQ ID NO: 3).

In a fourth aspect, the invention provides a gene OsGSL2 targeting vector developed based on a CRISPR/Cas9 system, wherein the targeting vector contains the sgRNA.

In a fifth aspect, the present invention provides a gene OsGSL2 mutant which is a mutant of wild type gene OsGSL2 wherein any base mutation of an exon causes amino acid mutation and/or protein dysfunction to thereby cause male sterile phenotype of rice.

Further, the mutant OsGSL2-3 is a mutant formed by deleting the 42 th to 60 th bases (5'-TTTGAGCAGAGACGTTTAT-3') from the initiation codon ATG of the wild-type gene OsGSL2 and deleting the 62 th base (A) of the complementary strand thereof.

The nucleotide sequence of the mutant OsGSL2-3 is shown as SEQ ID NO. 7, and the nucleotide sequence of the complementary strand is shown as SEQ ID NO. 8.

In a sixth aspect, the present invention provides a biological material comprising the mutant OsGSL2-3, the biological material including, but not limited to, recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, engineering bacteria or transgenic cell line.

In a seventh aspect, the present invention provides a method for preparing a male sterile line of rice, the method comprising: the rice male sterile line obtained by the method or the rice male sterile line containing the mutant OsGSL2-3 is used as a parent and hybridized with a target rice material, and the obtained F1 generation is backcrossed with the target rice material, so that the backcrossed offspring obtain the same characters and gene mutation as the rice male sterile line.

Preferably, the nucleotide sequence of the OsGSL2 gene in the rice male sterile line is shown as SEQ ID NO. 7, and the nucleotide sequence of the complementary strand is shown as SEQ ID NO. 8.

In an eighth aspect, the invention provides any one of the following uses of the sgRNA or a targeting vector containing the sgRNA or the mutant OsGSL2-3 or a biological material containing the mutant:

(1) Application in regulating male fertility character of rice;

(2) Application in rice crossbreeding and seed production;

(3) The application of the rice sterile line in breeding or germplasm resource improvement.

The crossbreeding and seed production means that an OsGSL2 male sterile line is used as a female parent to be hybridized with other male parents; the method comprises the steps of hybridizing the obtained OsGSL2 male sterile line with other target rice materials, obtaining F1 generation, and backcrossing with the target rice materials, so that the target materials obtain the character and gene mutation of the OsGSL2 male sterile line.

Such improvements include, but are not limited to, increasing crop yield, increasing crop quality, increasing crop pest resistance, stress resistance, and lodging resistance.

By means of the technical scheme, the invention has at least the following advantages and beneficial effects:

the invention discloses the biological functions of the OsGSL2 (LOC_Os01g 48200) gene and the coded protein for the first time, and the effect of the OsGSL2 gene on the aspect of regulating the male reproductive development of rice is not reported before. The invention utilizes CRISPR/Cas9 method to mutate rice gene OsGSL2, and discovers the regulation and control functions of the OsGSL2 gene on rice anther development. The method for editing CRISPR/Cas9 genes and the male sterile mutant obtained after editing can be used for creating a rice male sterile line, is used for rice cross breeding and seed production, and has important significance for researching rice male reproductive development.

Drawings

FIG. 1 is a block diagram of the OsGSL2 gene of the invention.

FIG. 2 is a diagram showing the expression pattern of the OsGSL2 gene of the present invention.

FIG. 3 is a schematic diagram of site-directed mutagenesis target sites in a preferred embodiment of the invention.

FIG. 4 shows T in a preferred embodiment of the invention ₀ Agarose gel electrophoresis diagram for detecting hygromycin of rice.

FIG. 5 shows T in a preferred embodiment of the invention ₀ Map of generation rice mutation types.

FIG. 6 is a schematic diagram of T in a preferred embodiment of the invention ₀ Generation of rice mutant sequencing peak patterns.

FIG. 7 shows a T-shape in accordance with a preferred embodiment of the present invention ₀ Anther morphology and pollen iodine staining pattern of the rice mutant.

FIG. 8 is a schematic diagram of a T-cell in accordance with a preferred embodiment of the present invention ₀ Generation of rice mutant spike figure.

FIG. 9 is a technical scheme of hybrid transformation of rice material according to the present invention. Wherein Osgls2 represents a male sterile line created by OsGSL2, and RP represents a conventional rice parent.

Detailed Description

The invention provides an application of a male sterile gene OsGSL2 and a mutant OsGSL2-3 thereof in creating a rice male sterile line.

The invention adopts the following technical scheme:

in a first aspect, the invention provides an application of an OsGSL2 gene in controlling male reproductive development of rice, wherein the amino acid sequence of a protein encoded by the OsGSL2 gene is any one of the following:

1) The amino acid sequence is shown as SEQ ID NO. 2;

2) The amino acid sequence is an amino acid sequence which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of SEQ ID NO. 2 and has the activity of regulating male fertility of plants.

The invention provides application of an OsGSL2 gene in controlling male reproductive development of rice, wherein the nucleotide sequence of the OsGSL2 gene is shown as SEQ ID NO. 1.

In a second aspect, the present invention provides a method of creating a male sterile line of rice, the method comprising: inhibiting the expression and/or activity of the OsGSL2 gene in rice plants.

In the method for creating the rice male sterile line, CRISPR/Cas9 gene editing is utilized to inhibit the expression and/or activity of the OsGSL2 gene.

The CRISPR/Cas9 gene editing comprises: and designing a CRISPR/Cas9 carrier target at the 1 st exon of the OsGSL2 gene, wherein the DNA sequence of the target is shown as SEQ ID NO. 3.

In a third aspect, the present invention provides a target site suitable for direct knockout of a plant OsGSL2 gene by a CRISPR/Cas9 system, the target site comprising: target site 1 (abbreviated as B1): 5'-GAGCAGAGACGTTTATAGAC-3' (SEQ ID NO: 3).

In a fourth aspect, the invention provides a CRISPR/Cas9 system targeting vector comprising an sgRNA specifically targeting said target site.

In a fifth aspect, the present invention provides a gene OsGSL2 mutant which is a type of nucleotide mutation causing amino acid mutation and/or protein dysfunction by any base mutation of wild-type OsGSL2 exon, thereby causing male sterile phenotype.

The mutant is a deletion mutant OsGSL2-1 of a wild OsGSL2 gene from 59 th base to 61 th base of an initiation codon ATG, and the sequence is shown as SEQ ID NO. 4; or the deletion of 59 th base to 63 th base from the ATG of the initiation codon of the wild OsGSL2 gene, and the deletion mutant OsGSL2-2 of 55 th to 64 th base of the complementary strand, the sequences of which are shown as SEQ ID NO. 5 and SEQ ID NO. 6; or the wild OsGSL2 gene is deleted from 42 th base to 60 th base from the start codon ATG, and the complementary strand 62 th base is deleted, and the sequences are shown as SEQ ID NO. 7 and SEQ ID NO. 8.

In a sixth aspect, the present invention provides a method for obtaining an OsGSL2 male sterile line, comprising: the rice male sterile line obtained by the method is used as a parent and hybridized with a target material, and the obtained F1 generation is backcrossed with the target material, so that the backcrossed offspring obtain the same character and gene mutation as those of the rice male sterile line.

Preferably, the nucleotide sequence of the OsGSL2 gene in the rice male sterile line is shown as SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 or SEQ ID NO. 8.

In a seventh aspect, the invention provides the use of a target site as defined above or an sgRNA as defined above or an OsGSL2 mutant as defined above in any of the following:

(1) Application in regulating male fertility character of rice;

(2) Application in rice crossbreeding and seed production;

(3) The application of the rice sterile line in breeding or germplasm resource improvement.

The crossbreeding and seed production means that an OsGSL2 male sterile line is used as a female parent to be hybridized with other male parents; the method comprises the steps of hybridizing the obtained OsGSL2 male sterile line with other target materials, obtaining F1 generation, and backcrossing with the target materials, so that the target materials obtain the character and gene mutation of the OsGSL2 male sterility.

The improvement comprises improving crop yield, improving crop quality, improving crop pest and disease resistance, stress resistance, lodging resistance and the like.

The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or in accordance with the manufacturer's instructions.

EXAMPLE 1 analysis of OsGSL2 Gene sequence and expression Pattern of Rice

In Ensembl Plants library (http:// Plants. Ensembl. Org/index. Html), a rice OsGSL2 (LOC_Os01g 48200) gene is queried, the nucleotide sequence in japonica rice is shown as SEQ ID NO.1, the gene function annotation is callose synthase (callose synthase), the coding protein of the gene comprises 469 amino acids, and the sequence is shown as SEQ ID NO. 2.

As the actual function of the OsGSL2 gene in rice has not been disclosed, in order to study the relation between the gene and the male reproductive development of rice, the invention firstly utilizes a gene expression database to analyze the expression patterns of the gene at different tissue parts of rice (figure 2), and the expression analysis result shows that the OsGSL2 gene is highly expressed in the spike stage of rice.

Example 2 function of Rice OsGSL2 Gene and creation of Rice Male sterile line Using CRISPR/Cas9 method

In order to determine the function of the rice OsGSL2 in rice, the invention adopts a CRISPR/Cas9 gene editing method to mutate the LOC_Os01g48200 gene sequence in a site-specific manner, and knock out the function of the gene in rice. The invention selects the flower 11 in the conventional rice of the rice as a receptor material for gene editing. The invention respectively selects the sequence shown in SEQ ID NO:3 from 45 th base to 64 th base of the coding region of the gene from the ATG of the initiation codon as a target region 1 (B1 for short, see figure 1) of CRISPR/Cas9 gene editing.

1. Construction of CRISPR/Cas9 gene editing vector of OsGSL2

The gene editing vector of the invention is pEGCas9Pubi-B-OsGSL2 (the vector map is shown in figure 3), and the basic vector of the vector is pEGCas9Pubi-B. The invention designs a target spot on a primer, obtains MT-sgRNA through PCR, and then connects the MT-sgRNA to a basic vector through a one-step cloning method, and the specific construction flow is as follows:

(1) Design of target gRNA. The gene sequence of OsGSL2 (LOC_Os01g 48200) is input into https:// zlab. Bio/guide-design-resources for target design, and the PAM sequence is set as NGG. The DNA sequence of the target area selected by the invention is shown as SEQ ID NO. 3.

(2) The sgRNA expression cassette was amplified by overlap PCR and nested PCR. Primer pairs containing the sgRNA target sequences are synthesized, annealed and then ligated with Bsa I digested binary vector pEGCas9Pubi-B (see Ma X, zhang Q, zhu Q.et al A Robust CRISPR/Cas9 System for Convenient, high-Efficiency Multiplex Genome Editing in Monocotand Dicot Plants, mol plant 2015,8 (8): 1274-1284, vector pEGCas9Pubi-B was given away by Hainan university Long Tuan teacher) to obtain recombinant vector pEGCas9Pubi-OsGSL2. The recombinant vector pEGCas9Pubi-OsGSL2 was transformed into escherichia coli dh5α, and positive clones were selected for sequencing, a method in specific step references (Xing, h.l., dong, l., wang, z.p., zhang, h.y., han, c.y., liu, b., wang, x.c., and Chen, q.j. (2014) a CRISPR/Cas9toolkit for multiplex genome editing in plants.bmc plant biology 14:327).

(3) And (5) sequencing and verification.

2. Agrobacterium-mediated genetic transformation of rice

Transferring the pEGCas9Pubi-OsGSL2 vector into agrobacterium EHA105 by a heat shock method, and adding glycerol to preserve bacterial liquid at-80 ℃ after PCR identification. Taking young embryos of flowers 11 in a freshly stripped rice hybrid of about 1.5mm as a receptor material, placing the stripped rice embryos into 2mL plastic centrifuge tubes containing 1.8mL suspension for not more than 1 hour, and placing about 100 young embryos into each centrifuge tube; the suspension was aspirated and the young embryos were rinsed 2 times with fresh suspension, the bottom of the tube remained a small amount of suspension that could have passed through the young embryos, then heat shock was applied at 43℃for 2 minutes, followed by an additional ice bath for 1 minute, the bottom residual wash was aspirated with a pipette, and 1.0mL of Agrobacterium infestation was added, gently shaken for 30 seconds, and then allowed to stand in the dark for 8 minutes. Pouring the young embryo and the infection liquid in the centrifuge tube into a co-culture medium, shaking uniformly, sucking out excessive infection liquid by using a pipetting gun, and co-culturing in darkness at 23 ℃ for 3 days with scutellum of all young embryos facing upwards. After the co-cultivation is finished, the young embryo is transferred to a recovery culture medium by sterile forceps, and is cultivated for 7-14 days at 28 ℃, and the young embryo growing on the young embryo needs to be removed in time in the middle process. After the recovery culture, the young embryo is placed on 1.5mg/L biamphos screening medium for screening and culturing for 3 rounds, each round of screening for 2 weeks, and then transferred to 2mg/L biamphos screening medium for screening and culturing for 2 rounds, and each round of screening for 2 weeks. The resistant calli were transferred to expansion medium and dark cultured for 2 weeks at 28 ℃. The propagated resistant calli were then transferred to induction medium and incubated for 2 weeks at 28℃in the dark. Then transferred to a differentiation medium, cultured at 25℃and 5000lx under light for 2 weeks. After the cultivation is finished, single seedlings are separated from the differentiated seedling clusters and placed in a rooting medium, and the seedlings are subjected to illumination cultivation at 25 ℃ and 5000lx until rooting; transferring the young seedling into a small nutrition pot for growth, transplanting the young seedling into a greenhouse after the young seedling survives growth, and harvesting offspring seeds after 3-4 months.

3、T ₀ CRISPR/Cas9 mutation result detection of generation plants

To determine T ₀ The CRISPR/Cas9 mutation result of the generation plant is carried out by adopting the following steps:

the invention firstly adopts a CTAB method to extract rice leaf DNA, and the specific method is as follows: the DNA extraction method was performed with a slight improvement over the conventional CTAB method (Rogers and Bendich, 1985). Placing 3cm rice leaf into a sterilized 2mL centrifuge tube, adding 6mm steel ball, performing tissue disruption with cell disruption instrument, performing CTAB extraction, and adding 200 μl of sterilized water (ddH) ₂ O), dissolving the air-dried sample DNA for later use. After complete DNA solubilization, 2. Mu.L of the sample was aspirated and the OD (A260/A280) and concentration of nucleic acid was determined using an ultraviolet spectrophotometer (Nanodrop 2000) and the DNA sample was diluted to 50 ng/. Mu.L for use.

Positive detection primer: hn-F and Hn-R; product size: 561bp; the primer sequences were as follows:

Hn-F:5’-CTTAGCCAGACGAGCGGGTTC-3’

Hn-R:5’-GCTTCTGCGGGCGATTTGT-3’

the positive detection result is shown in FIG. 4, negative control H ₂ O and ZH11 are free of bands, positive plasmid is controlled to amplify 561bp fragment, T ₀ The detection results of the generation plants are positive, so that the genotypes can be further detected. Amplification and detection according to the following PCR parametersMeasuring the genotype of the mutant:

PCR was performed using a2 XPCR premix of Biomiga (Mg-containing ²⁺ The method comprises the steps of carrying out a first treatment on the surface of the Taq DNA Polymerase;2.5mM dNTPs;10 XPCR Buffer) 5. Mu.L, 1. Mu.L primer (10. Mu. Mol/L forward and reverse primers each 0.5. Mu.L), 1. Mu.L template DNA, ddH ₂ O makes up 10. Mu.L. The PCR amplification procedure was a conventional SSR procedure (pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 55℃for 30s, extension at 72℃for 30s, amplification for 35 cycles; extension at 72℃for 5 min). The amplified product was subjected to 6% non-denaturing polyacrylamide gel electrophoresis, 0.1% AgNO ₃ After dyeing, formaldehyde and NaOH chromogenic photographing, genotype statistics is carried out, and finally 3T's are found ₀ The sequence of the transformation event target region is changed, the phenotype is different, the sequence before and after editing is shown in figure 5, corresponding to 3 OsGSL2 mutants: osgsl2-1, osgsl2-2 and osgsl2-3.

Comparing the nucleotide sequences in 3 OsGSL2 mutants (figure 5 and figure 6), compared with the unedited WT, the mutated strains OsGSL2-1, osGSL2-2 and OsGSL2-3 have the deletion of the coded nucleotide at the target, the OsGSL2-1 has the deletion of the 59 th base to the 61 th ATA base from the start codon ATG, and the sequence is shown as SEQ ID NO. 4; the osgsl2-2 is deleted from the 59 th base to the 63 th base of the ATG of the initiation codon, and the 55 th base to the 64 th base of the complementary strand GTTTATAGAC is deleted, and the sequences are shown as SEQ ID NO. 5 and SEQ ID NO. 6; the osgsl2-3 is deleted from the 42 th base to the 60 th base TTTGAGCAGAGACGTTTAT from the ATG of the initiation codon, and the base A of the complementary strand 62 is deleted, and the sequences are shown as SEQ ID NO. 7 and SEQ ID NO. 8.

Deletion of the nucleotides encoded in the mutated lines osgsl2-1, osgsl2-2 and osgsl2-3 results in a frame shift of the amino acids and in premature termination of the amino acid translation. Thus, the LOC_Os01g48200 protein functions of the transformants were deleted.

EXAMPLE 3 phenotypic analysis of OsGSL2 sterile line

Observation of tassel, anther and pollen viability of OsGSL2 sterile line: plants of the OsGSL2 sterile lines (OsGSL 2-1, osGSL2-2 and OsGSL 2-3) were substantially unchanged from the wild type in terms of vegetative growth and tassel development; wild in tassel developmentThe male can be normally pulled out, anthers can be normally cracked and loose powder, the anthers can be normally firm after selfing, while the OsGSL2 sterile line can be normally pulled out and can also be normally opened, but the OsGSL2-1 anthers are shriveled, white and slightly green, the OsGSL2-2 and the OsGSL2-3 anthers are shriveled, slightly white (figure 7); further performing I on wild type and mutant pollen ₂ KI staining, normal development of wild type pollen, black after pollen grain staining, while 99% of the mutant osgsl2-1 pollen grains failed to stain, 100% of the osgsl2-2 pollen grains failed to stain, osgsl2-3 no pollen grains (fig. 7). The seed setting rate is counted by taking the snapping seeds in the mature period, the wild ZH11 seed setting is normal, and the seed setting rate is more than 90%; the selfing setting rate of the mutant osgsl2-1 is about 1%; the selfing rates of the mutants osgsl2-2 and osgsl2-3 were about 0% (FIG. 8). This suggests that the OsGSL2 (LOC_Os01g 48200) gene controls male development of rice by affecting male gamete development of anthers.

Example 4 sterile line transfer of OsGSL2 mutant Gene

Hybridization, backcrossing and selfing are carried out by using the OsGSL2 mutant and receptors with normal fertility, such as Bo II B and wild incense B, and molecular markers are used for carrying out OsGSL2 gene and genetic prospect selection in the process, so that the recessive nuclear sterile line with homozygous OsGSL2 mutant genes under the background of Bo II B and wild incense B is finally obtained. The technical route of hybridization transformation is shown in fig. 9, and the specific operation steps are as follows:

1. hybridization of acceptor parents, such as Bo II B and wild incense B, as male parent and female parent OsGSL2 to obtain F ₁ 。

2. Backcrossing F1 as female parent and receptor parent such as Bo II B and Lemongrass B to obtain BC ₁ F ₁ 。

3. Planting BC ₁ F ₁ Detecting the genotype of the OsGSL2 by using primer pairs with primer sequences shown as SEQ ID No.11-12 respectively, and selecting the heterozygous genotype of the OsGSL2, namely, simultaneously generating 233bp and 236bp bands of PCR amplified products.

4. And (3) carrying out genetic background identification on the single plants selected in the step (3) by using a group of genotypes (such as 200) with polymorphism between the OsGSL2 mutant and recurrent parent and uniformly distributed molecular markers (including but not limited to SSR, SNP, INDEL, EST, RFLP, AFLP, RAPD, SCAR type markers), and selecting plants with high similarity (such as more than 88% similarity or 2% medium selection rate) with the recurrent parent genotypes.

5. Backcrossing the plant selected in step 4 with recipient parents, such as Bo II B and Ledebouriella sessilifolia B, to obtain BC ₂ F ₁ 。

6. Planting BC ₂ F ₁ Repeating the steps 3 and 4, selecting plants with high recovery rate (such as more than 98% or 2% selection rate) of the genetic background by heterozygous OsGSL2 genotype, and collecting the selfing seeds BC ₂ F ₂ 。

7. Planting BC ₂ F ₂ Repeating the step 3 and the step 4, selecting plants with highest heterozygosity of the OsGSL2 genotype and the genetic background homozygous rate, and collecting the inbred seeds BC ₂ F ₃ 。BC ₂ F ₃ The homozygous strain of the OsGSL2 separated in the offspring is the sterile line of the OsGSL2 gene.

The above uses Bo II B and wild incense B as transformation examples, but is not limited to Bo II B and wild incense B, and can be any rice material.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims (10)

1. The application of the gene OsGSL2 in the regulation of male fertility of rice is characterized in that the gene OsGSL2 is a gene encoding the following protein (a) or (b):

(a) A protein consisting of the amino acid sequence shown in SEQ ID NO. 2; or (b)

(b) A protein derived from (a) with equivalent functions and with the sequence shown in SEQ ID NO. 2 being substituted, deleted or added with one or more amino acids;

the gene OsGSL2 is modified to ensure that the gene is in a loss or weakening state, so that the rice obtains the male sterile character.

2. A method of creating a male sterile line of rice, the method comprising: inhibiting the expression and/or activity of gene OsGSL2 in rice plants;

wherein the gene OsGSL2 is the same as the gene as described in claim 1.

3. The method according to claim 2, wherein the means for inhibiting the expression and/or activity of gene OsGSL2 comprises a gene editing technique or introducing a molecule having the expression and/or activity of gene OsGSL2 into rice;

the molecule with the gene OsGSL2 expression and/or activity is at least one selected from shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA/DNA, low molecular compounds, peptides and antibodies;

the gene editing techniques include CRISPR, TALEN, and ZFN.

4. The method of claim 3, wherein CRISPR/Cas9 gene editing is utilized to inhibit the expression and/or activity of an OsGSL2 gene.

5. The CRISPR/Cas9 system-based targeting gene OsGSL2 sgRNA is characterized in that the sgRNA targets the 1 st exon of the gene OsGSL2, and the nucleotide sequence of the action site is 5'-GAGCAGAGACGTTTATAGACTGG-3';

wherein the gene OsGSL2 is the same as the gene as described in claim 1.

6. A gene OsGSL2 targeting vector developed based on a CRISPR/Cas9 system, characterized in that the targeting vector contains the sgRNA of claim 5;

wherein the gene OsGSL2 is the same as the gene as described in claim 1.

7. The gene OsGSL2 mutant is characterized in that the gene OsGSL2 mutant is a mutant with amino acid mutation and/or abnormal protein function caused by random base mutation of an exon of a wild gene OsGSL2, thereby causing a rice male sterile phenotype.

8. The mutant according to claim 7, which is a mutant OsGSL2-3, wherein the wild-type gene OsGSL2 is obtained by deleting the 42 th to 60 th bases from the initiation codon ATG and deleting the 62 th bases of the complementary strand;

the nucleotide sequence of the mutant OsGSL2-3 is shown as SEQ ID NO. 7, and the nucleotide sequence of the complementary strand is shown as SEQ ID NO. 8.

9. The preparation method of the male sterile line of the rice is characterized by comprising the following steps: taking the rice male sterile line obtained by the method of any one of claims 2-4 or the rice male sterile line containing the mutant OsGSL2-3 of claim 7 or 8 as a parent, hybridizing with a target rice material, and backcrossing the obtained F1 generation with the target rice material, so that the backcrossed offspring obtain the same characters and gene mutation as the rice male sterile line;

preferably, the nucleotide sequence of the OsGSL2 gene in the rice male sterile line is shown as SEQ ID NO. 7, and the nucleotide sequence of the complementary strand is shown as SEQ ID NO. 8.

10. Use of the sgRNA of claim 5 or the targeting vector of claim 6 or any of the following of the mutant OsGSL2-3 of claim 7 or 8:

(1) Application in regulating male fertility character of rice;

(2) Application in rice crossbreeding and seed production;

(3) The application of the rice sterile line in breeding or germplasm resource improvement.