Application of protein OsCYP89C2 or biological material in regulation and control of rice anther development

Abstract

The invention provides an application of a protein OsCYP89C2 or a biological material in regulation and control of rice anther development. Wherein the amino acid sequence of the protein OsCYP89C2 is as shown in SEQ ID NO: 1. The biological material comprises a nucleic acid molecule for coding the OsCYP89C2 protein, an expression cassette, a recombinant vector, a transgenic plant cell line, a transgenic plant tissue, a transgenic plant organ or a recombinant microorganism. The plant phenotype related protein OsCYP89C2 and the coding gene thereof provided by the invention can be used for regulating and controlling the plant phenotype, specifically, the knockout mutant oscy89c2 shows that the fruiting rate of ears is reduced, and part of anther develops abnormally; the fertility of a mutant plant into which a genome complementary fragment is transferred is recovered, and the pollen staining and the maturing rate are not obviously different from those of a wild type.

Description

[0001] field of study

[0002] The invention relates to the field of plant genetic engineering, and in particular to application of a protein OsCYP89C2 or a biological material in regulating the development of rice anthers.

[0003] Background

[0004] Rice belongs to the Poaceae family. Its inflorescence belongs to a panicle with an inflorescence axis. The first inflorescence branch and the secondary inflorescence branch are produced on the inflorescence axis, and the spikelets are distributed on these branches. Among them, the spikelets are composed of guard lemma, inner and outer palea, lodicules, stamens and pistils. The stamens include two structures, anthers and filaments. The anthers contain two pollen sacs, and each pollen sac contains two anther chambers. The wall of each anther chamber is composed of 4 different cells, from the inside to the outside, respectively, tapetum cells, middle layer cells, endodermal cells and epidermal cells, and these cells are differentiated from the L1 and L2 layer cells of the flower primordium. The L1 layer cells differentiate into epidermal cells, and the L2 layer cells differentiate into the other three layers of cells. With the development of microspores, the tapetum, middle layer and endodermal cells will degrade. The surface of the anther is covered with a waxy layer and a cuticle, which play a supporting and protective role for the anther.

[0005] Anther development is a very complex biological process, including cell differentiation, meiosis, development and maturation of microspores, and the shedding of pollen grains, which can be divided into 14 developmental stages. The first to sixth stages of anther development are mainly cell differentiation: including the formation of stamen primordium, differentiation of stamen primordium, spore cell division and differentiation into primary cell wall cells, archosporocyte division to form primary sporangioblasts, and primary wall cells forming two secondary cell wall cell layers, primary sporangioblasts dividing and differentiating into sporangioblasts, and secondary wall cells differentiating to form endodermis, middle layer and tapetum, and secondary sporangioblasts differentiating to form microspore mother cells. The seventh and eighth stages of anther development are mainly microspore mother cells undergoing meiosis: including meiosis to form tetrads and the successive degeneration of the tapetum. The 9th to 12th stages of anther development are mainly the development and maturation of microspores: the tapetum secretes callose, dissolving the callose wall and releasing microspores; the tapetum continues to degrade, the number of Wulbricht bodies increases, the microspores become vacuolated and rounded, and the microspore outer wall forms a thicker outer wall; the microspore undergoes the first mitosis and asymmetric cell division to produce a small reproductive cell and a large nutrient cell. At this time, the microspore becomes sickle-shaped and the tapetum is almost completely degraded; the microspore undergoes the second mitosis to produce two male gametes, and the mature pollen grains contain 3 nuclei and accumulate a large amount of starch and lipid substances. The 13th and 14th stages of anther development are mainly the shedding of pollen grains: including pollen rounding, opening of inner and outer lemmas, anther dehiscence, and rupture of septa.

[0006] At present, several genes of rice male sterile mutants have been cloned. These genes act at different stages of anther development and play different functions in the process of anther development. According to their functions in regulating rice anther development, these genes can be classified into three categories: genes involved in early anther regulation, genes involved in meiosis, and genes involved in late anther development regulation. Among them, the cytochrome P450 superfamily that has been reported to be involved in rice anther development includes CYP703 and CYP704. CYP703A3 is expressed in the tapetum from period 8b to period 10, and is relatively weakly expressed in microspores. This gene encodes a fatty acid ω-hydroxylase. CYP703A3 can catalyze lauric acid to produce 7-hydroxylauric acid, which is involved in the formation of sporopollenin precursors. The anthers of the cyp703a3-2 mutant are thin and white, and the pollen is powderless. Transmission electron microscopy results showed that the mutant pollen exine was lightly stained, indicating abnormal deposition of sporopollenin; the mutant had fewer Wulbricht bodies. Wulbricht bodies can transport sporopollenin precursors from the tapetum to the outer surface of the microspores. Chemical analysis showed that the cutin monomers and wax content of the mutant anthers were reduced. These results indicate that the gene is involved in the formation of the pollen exine and anther epidermis. CYP704B2 is expressed in the tapetum and microspores from the 8th to the 10th period of anther development, catalyzing the ω-hydroxylation of carbon chains with 16 and 18 carbons. After CYP704B2 mutation, its anthers are thin and white, and the pollen is powderless; the tapetum degradation is abnormal, the pollen exine development is defective, and the anther cuticle is rapidly degraded. Chemical composition analysis found that the mutant has fewer cutin monomers. These results indicate that CYP704B2 is involved in the biosynthesis of sporopollenin and cutin.

[0007] At present, there are few reports on the functional research of CYP89 family genes, and no research related to fertility has been found. Rice male fertility is closely related to breeding. Therefore, further research on fertility-related genes will help improve the molecular regulatory mechanism of rice anther development and help obtain new plant varieties with excellent traits that can be stably inherited through molecular breeding. Summary of the invention

[0008] The main purpose of the present invention is to provide an application of protein OsCYP89C2 protein or biological material in regulating rice anther development, so as to supplement and improve the rice male sterile mutant gene system known in the prior art.

[0009] In order to achieve the above object, according to the first aspect of the present invention, a protein OsCYP89C2 or a biomaterial is provided for use in regulating the development of rice anthers. The amino acid sequence of the protein regulating the development of rice anthers described in the present invention is shown in SEQ ID NO: 1 in the sequence list, and is composed of 517 amino acid residues. The protein of the oscyp89c2 mutant undergoes a frameshift mutation after the 136th amino acid, and the mutant protein sequence is shown in SEQ ID NO: 4 in the sequence list.

[0010] The biological material of the present invention comprises any one of the following a)-g): a) a nucleic acid molecule encoding an OsCYP89C2 protein; b) an expression cassette containing the nucleic acid molecule; c) a recombinant vector containing the nucleic acid molecule or the expression cassette; d) a recombinant microorganism containing the nucleic acid molecule, the expression cassette or the recombinant vector; e) a transgenic plant cell line containing the nucleic acid molecule or the expression cassette; f) a transgenic plant tissue containing the nucleic acid molecule or the expression cassette; g) a transgenic plant organ containing the nucleic acid molecule or the expression cassette.

[0011] The gene encoding the above protein may be the cDNA sequence of the gene, such as the nucleotide sequence shown in SEQ ID NO: 2 in the sequence list. In the oscyp89c2 mutant, the 409th base guanine (G) is missing, and it may also be the genomic DNA sequence of the gene, such as the nucleotide sequence shown in SEQ ID NO: 3 in the sequence list.

[0012] In order to achieve the above object, according to the second aspect of the present invention, a method for obtaining a rice anther development abnormality or male partial sterility mutant is provided. The OsCYP89C2 gene is site-directed edited by CRISPR / Cas9 gene editing to obtain the oscyp89c2 mutant. The mutation occurs at the 409th base after ATG, and the base G is missing, resulting in a protein frameshift mutation.

[0013] In order to achieve the above object, according to the third aspect of the present invention, a method for restoring abnormal anther development or male partial sterility in rice is provided. The steps include: constructing a binary vector containing the OsCYP89C2 gene promoter and gene region, and introducing it into the oscyp89c2 mutant through genetic transformation, thereby obtaining a plant with restored fertility.

[0014] By applying the technical solution of the present invention, the expression level of the OsCYP89C2 protein or gene in the plant can be regulated to achieve the regulation of rice anther fertility and obtain a partially sterile mutant. This can be edited together with other genes to obtain a more thorough male sterile mutant, which can be used in rice breeding and is of great significance to rice breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of phenotypic analysis of wild type and oscyp89c2 mutant.

[0016] Figure 2 Schematic diagram of phenotypic analysis of gene complementation. DETAILED DESCRIPTION

[0017] The present invention is further described below in conjunction with specific embodiments for better understanding, but the scope of the present invention is not limited thereby.

[0018] Example 1: Gene editing CRISPR / Cas9 to create oscyp89c2 mutant

[0019] OsCYP89C2 was gene-edited by CRISPR / Cas9, and two specific CRISPR knockout target sites (the 3′ end 3 bases were PAM sequences) were designed for the target gene on the CRISPR-GE website (http: / / skl.scau.edu.cn / ), and the sequence was as follows: 5′-GCCCTCCACGTGGGGCCATA-3′.

[0020] The gene knockout vector was constructed using the pYLCRISPR / Cas9-MH vector provided by Professor Liu Yaoguang's research group at South China Agricultural University. The specific construction steps were carried out according to the supporting operation guide provided by the research group. The designed target plus the sequence complementary to the sticky end of the backbone vector was used as the primer of the target, and the primer of the knockout target was synthesized by BGI; then the pYLCRISPR / Cas9-MH and gRNA plasmids were treated with BsaⅠ endonuclease to verify whether the plasmids were normal, and then the designed knockout primers were used to make the target linker; then the target linker and the gRNA expression cassette were connected together by cutting and connecting with BsaⅠ endonuclease and T4 DNA ligase, and then amplified by two rounds of PCR and detected the target band by gel electrophoresis, cut the gel and recovered, and detected the concentration of the recovered product; then the recovered target fragment was connected to the pYLCRISPR / Cas9-MH knockout vector with BsaⅠ endonuclease and T4 DNA ligase, and the ligation product was heat-shocked and transformed into Escherichia coli, and the colonies of positive clones were selected and shaken to extract the plasmid for sequencing. The correctly sequenced plasmid was transferred into Agl0 Agrobacterium, and then introduced into callus tissue through genetic transformation of rice for tissue culture to obtain the corresponding transgenic seedlings.

[0021] Molecular identification and sequencing analysis were performed on these transgenic seedlings to obtain the mutant oscyp89c2 of OsCYP89C2, whose mutation occurred at the 409th base after ATG, with the base guanine (G) missing, resulting in a frameshift mutation in the protein. The mutant nucleotide sequence is shown in SEQ ID NO:5 in the sequence listing.

[0022] Example 2: Phenotypic analysis of oscyp89c2 mutant

[0023] The oscyp89c2 mutant showed no significant difference in vegetative growth compared with the wild type ( Figure 1 A), but the ear set decreased ( Figure 1 B), the fruiting rate of the mutant is only about 50%, which is significantly different from that of the wild type ( Figure 1 C). Only some of the mutant florets have normal anther morphology, while most of the florets have white and thin anthers to varying degrees. ( Figure 1 D and 1E). Pollen potassium iodide staining results showed that some florets of the mutant had normal pollen staining, while most florets had only a few that could be stained, and most of them were abortive ( Figure 1 The above results indicate that the mutation of OsCYP89C2 affects the male fertility of rice.

[0024] In order to determine whether the female fertility of the mutant is normal, oscyp89c2 and osnp1, a male sterile mutant with normal female fertility, were used as female parents and Wuyunjing as male parents for hybridization verification, with osnp1 as the control. The hybridization results showed that the hybrid fruiting of oscyp89c2 was not significantly different from that of osnp1 ( Figure 1 H), indicating that the fertility of oscyp89c2 females is normal and the reason for the decrease in fruit set is due to the partial infertility of males.

[0025] Example 3: Gene complementation to restore oscyp89c2 mutation virulence

[0026] The function of OsCYP89C2 gene was further verified by gene function complementation experiment. Primers were designed to amplify OsCYP89C2 gene, and complementary vector was constructed, including 2602bp upstream of OsCYP89C2, 1548bp coding region (removing terminator), nucleotide sequence as shown in SEQ ID NO:6 in the sequence table, and 720bp GFP (nucleotide sequence as shown in SEQ ID NO:7), which was transferred to oscyp89c2 mutant. The fragment was connected to pCAMBIA1300 vector by In-fusion HD Cloning homology arm recombination method, and the recombinant plasmid was transformed into Escherichia coli competent DH5α. After positive detection and sequencing identification, the obtained genomic complementary vector was named pCAMBIA1300-OsCYP89C2-GFP. Subsequently, the plasmid was transferred into Agrobacterium, and the target fragment was introduced into the callus tissue of oscyp89c2 mutant seeds by Agrobacterium-mediated. From the transgenic positive plants obtained, the genomic background of the plants was determined to be homozygous for oscyp89c2 mutation, and the phenotypes of their offspring were examined. The results showed that two transgenic positive plants of oscyp89c2 mutant (1 st -1-8 and 2 nd -2-7) are completely dyeable with potassium iodide staining ( Figure 2 A), indicating that the fertility of pollen grains has been restored; the fruiting rate of transgenic positive plants of oscyp89c2 mutant is significantly higher than that of mutant, and has no difference with wild type ( Figure 2 B) The experiment showed that the fertility of the oscyp89c2 mutant can be restored by gene complementation, and the OsCYP89C2 gene has the function of regulating the development of rice anthers.

Claims

1. Application of OsCYP89C2 protein or biological material in regulating rice anther development, characterized in that: The amino acid sequence of the OsCYP89C2 protein is shown in SEQ ID NO: 1; the biological material comprises any one of the following a)-g): a) a nucleic acid molecule encoding an OsCYP89C2 protein; b) an expression cassette containing the nucleic acid molecule; c) a recombinant vector containing the nucleic acid molecule or the expression cassette; d) a recombinant microorganism containing the nucleic acid molecule, the expression cassette or the recombinant vector; e) a transgenic plant cell line containing said nucleic acid molecule or said expression cassette; f) transgenic plant tissue containing the nucleic acid molecule or the expression cassette; g) a transgenic plant organ containing the nucleic acid molecule or the expression cassette.

2. The use according to claim 1, characterized in that: Mutation of the protein OsCYP89C2 can lead to partial male sterility phenotype in rice.

3. The use according to claim 2, characterized in that: The amino acid sequence of the mutant protein OsCYP89C2 is shown in SEQ ID NO: 1, and the gene sequence encoding the mutant protein is shown in SEQ ID NO: 2 or 3.

4. A method for obtaining a rice mutant with abnormal anther development or partial male sterility, characterized in that: include: Using genetic engineering to knock out OsCYP89C2 Gene; among them, OsCYP89C2 The gene sequence is shown in SEQ ID NO: 2 or 3.

5. The method according to claim 4, characterized in that The genetic engineering method is to OsCYP89C2 The sgRNA sequence based on CRISPR / Cas9 is designed as the target, the DNA fragment containing the sgRNA sequence is connected to the vector carrying CRISPR / Cas9, and the rice is transformed to obtain the transgenic rice with the gene function missing.

6. The method according to claim 5, characterized in that The nucleotide sequence of the sgRNA action site is 5'-GCCCTCCACGTGGGGCCATA-3'.

7. A method for restoring abnormal anther development or male partial sterility in rice, characterized in that: include: Will OsCYP89C2 The promoter and gene region sequence of were introduced into the mutant by genetic engineering; OsCYP89C2 The gene sequence is shown in SEQ ID NO: 2 or 3.

8. The method according to claim 6, characterized in that The genetic engineering method is to OsCYP89C2 The promoter and its genome were constructed into a plant binary expression vector and transformed into rice to obtain transgenic plants that complemented the mutant phenotype of the gene.

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