Gene OsCYP86A9 for regulating and controlling development of rice anther as well as encoding protein and application of gene OsCYP86A9

Abstract

The invention relates to a gene OsCYP86A9 for regulating and controlling rice anther development as well as an encoding protein and application thereof. The amino acid sequence and the nucleotide sequence of the rice anther development regulation gene OsCYP86A9 are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2 or SEQ ID NO. 3. The gene OsCYP86A9 and the encoding protein thereof provided by the invention can be used for regulating and controlling the fertility and the maturing rate of the rice pollen: the phenotype of a gene knockout plant CR-1 is consistent with that of an EMS mutant oscy86a9, and the phenotype of the gene knockout plant CR-1 and the phenotype of the EMS mutant oscy86a9 both show that the maturing rate of ears is reduced, and part of pollen is aborted; the fertility of a mutant plant into which a genome complementary fragment is transferred is recovered, and the anther form, pollen staining and the maturing rate are not obviously different from those of a wild type; the mutation of the OsCYP86A9 affects the programmed death of the anther tapetum and the formation of the outer wall and the inner layer of the pollen.

Description

[0001] field of study

[0002] The invention relates to the field of plant genetic engineering, and in particular to a gene related to rice anther development, a coded protein and an application thereof.

[0003] Background

[0004] Rice male sterility refers to a phenomenon in which pollen, i.e. microspores, develop abnormally while pistils develop normally. Anther development is a very complex biological process, including cell differentiation, meiosis, development and maturation of microspores, and the shedding of pollen grains. In this process, the formation of the pollen wall has far-reaching significance. The pollen wall is the outermost cell wall in the pollen grain, which is composed of the outer layer of the pollen exine and the inner layer of the pollen exine. The main component of the pollen wall is sporopollenin, which is an extremely strong substance that plays an important role in protecting male gametes from interference from various external environments.

[0005] At present, biochemical and genetic analysis studies have found that many genes related to fatty acid metabolism in plants are involved in the formation of pollen walls. DPW (defective pollen wall) encodes fatty acid reductase, which can catalyze acyl carrier proteins (ACPs) to produce hexadecanol, which is very important for the synthesis of primary fatty alcohols. It is expressed in both the tapetum and microspores. The dpw mutant showed thin anthers, white color, and pollen without powder; abnormal tapetum degradation, defective development of the outer and inner layers of the pollen exine, and finally complete degradation of microspores. WDA1 (Wax-deficient anther1) encodes an enzyme involved in the decarbonylation pathway of alkane biosynthesis. The mutant with T-DNA insertion showed thin anthers, white color, and pollen without powder. This mutant has defects in the biosynthesis of long-chain fatty acids. ACOS5 encodes acyl-CoA synthetase 5, which is located in the cytoplasm and is essential for the formation of pollen walls. OsACOS12 can catalyze C18:1 fatty acids to form C18:1-CoA, which is involved in the formation of cutin, wax and sporopollenin precursors (Li et al., 2016). ACOS5, OsACOS12 and PpACOS6 are homologous genes, and the expression of these three genes is directly regulated by the MYB transcription factor MSS188.

[0006] Among cytochrome P450, CYP703A and CYP704B are two families that hydroxylate fatty acids and are essential for sporopollenin formation. CYP703A3 encodes a fatty acid hydroxylase that only catalyzes the hydroxylation of lauric acid to produce 7-hydroxylauric acid. After mutation, this gene exhibits pollen wall defects, and the expression levels of genes involved in sporopollenin synthesis and transport are all reduced in the cyp703a3-3 mutant. The expression of the CYP703A3 gene is regulated by OsMS188 / OsMYB80, and the CYP703A3 promoter can bind to GAMYB and TDR (a gene that regulates programmed cell death in the tapetum and participates in the formation of pollen exine), which indicates that CYP703A3 is essential for the development of rice pollen exine. CYP703A2 can catalyze the conversion of medium-chain saturated fatty acids into the corresponding monohydroxy fatty acids, preferentially hydroxylating lauric acid to produce 7-hydroxylauric acid. This gene is expressed in both the tapetum and microspores. Mutations in this gene result in impaired pollen development, partial male sterility, lack of exine in the pollen wall, and lack of phenylpropanoid monomers in sporopollenin in CYP703A2 knockout mutants. These results suggest that hydroxylation within the lauric acid chain is essential for the synthesis of sporopollenin and phenylpropanoids. CYP704B1 is specifically expressed in anthers and is essential for the development of the pollen exine. cyp704b1 exhibits partial male sterility, damaged pollen wall, and lack of a normal exine layer. Heterologous expression in yeast revealed that CYP704B1 can catalyze the ω-hydroxylation of long-chain fatty acids, indicating that these fatty acids are involved in the formation of the pollen exine. CYP704B2 can catalyze the ω-hydroxylation of C16 and C18 fatty acids. cyp704b2 exhibits tapetum swelling, pollen abortion, and no exine. In addition, the expression of CYP704B2 gene was regulated by OsMS188 / OsMYB80.

[0007] Anther development is closely related to male sterility, and the gene of male sterility is of great significance in rice hybrid breeding. Therefore, further separation of genes controlling rice fertility not only provides a reference for clarifying the molecular regulatory mechanism of anther development, but also helps to cultivate new rice varieties through molecular breeding methods. At present, few cytochrome P450 genes involved in rice anther development have been reported, so the molecular regulatory mechanism of cytochrome P450 genes in the reproductive development process can also be further improved. Summary of the invention

[0008] The main purpose of the present invention is to provide a gene OsCYP86A9 for regulating rice anther development and its encoded protein and application, so as to solve the problem of the lack of such fertility proteins in the prior art.

[0009] To achieve the above object, according to a first aspect of the present invention, there is provided a protein OsCYP86A9 for regulating rice anther development, which is any one of the following (a)-(c): (a) a protein having an amino acid sequence as shown in SEQ ID NO: 1; or (b) a protein having the function of regulating rice anther development after substitution and / or deletion and / or addition of one or more amino acids in the amino acid sequence in (a); or (c) a protein having more than 80% homology with the amino acid sequence defined in any one of (a) and (b) and having the same function.

[0010] In order to achieve the above object, according to the second aspect of the present invention, a gene for regulating rice anther development is provided, which is any one of the following (a)-(d): (a) a nucleotide sequence encoding the protein OsCYP86A9 for regulating rice anther development as claimed in claim 1; or (b) a nucleotide sequence that hybridizes with the DNA molecule defined in (a) under stringent conditions and encodes the protein OsCYP86A9 for regulating rice anther development as claimed in claim 1; or (c) a nucleotide sequence as shown in SEQ ID NO: 2 or 3; or (d) a gene having more than 70% homology with any one of the nucleotide sequences defined in (a)-(c) and encoding a protein with the same function.

[0011] In order to achieve the above object, according to the third aspect of the present invention, a rice partial male sterility mutant is provided, named oscyp86a9, the male sterility of which is caused by abnormal tapetum programmed death and defective microspore outer wall development. Genetic analysis shows that the mutation is a recessive mutation controlled by a single gene. The inventors located the mutant gene by high-throughput sequencing and SIMM analysis using Illumina Hiseq 2000 platform. The mutation is caused by the mutation of the 941st base in the coding region of the OsCYP86A9 gene from guanine (G) to adenine (A), resulting in the mutation of the 314th amino acid of its encoded protein from arginine (Arg) to histidine (His). The nucleic acid sequence is shown in SEQ ID NO: 5, and the amino acid sequence is shown in SEQ ID NO: 4.

[0012] The amino acid sequence of the wild-type rice anther development-related protein OsCYP86A9 provided by the present invention is shown in SEQ ID NO: 1, and the protein sequence consists of 545 amino acid residues. In the oscyp86a9 mutant, the 314th amino acid of the protein is mutated from arginine (Arg) of the wild type to histidine (His), and the amino acid sequence is shown in SEQ ID NO: 4.

[0013] The gene encoding the wild-type protein may be the cDNA sequence of the gene, as shown in SEQ ID NO: 2. In the oscyp86a9 mutant, the 941st base is mutated from wild-type guanine (G) to adenine (A), or may be the genomic DNA sequence of the gene, the nucleotide sequence of which is shown in SEQ ID NO: 3.

[0014] In order to achieve the above object, according to a fourth aspect of the present invention, a method for obtaining a rice anther development abnormal mutant is provided, wherein the OsCYP86A9 gene is site-directedly edited by CRISPR / Cas9 gene editing to obtain a new allelic mutant.

[0015] In order to achieve the above object, according to a fifth aspect of the present invention, a method for restoring the abnormal anther development phenotype of rice is provided. The steps include: constructing a binary vector containing the OsCYP86A9 gene promoter and its gene region sequence, and introducing it into the oscyp86a9 mutant by genetic transformation, thereby obtaining a plant with a normal phenotype.

[0016] The anther development regulating gene OsCYP86A9 provided by the present invention affects anther development after mutation: abnormal tapetum degradation and rupture of the inner layer of the pollen outer wall lead to abnormal anther development and a significant decrease in the plant fruit setting rate.

[0017] The technical solution of the present invention provides a new protein for regulating rice anther development and its encoding gene and application, which is helpful for analyzing the research on the regulatory mechanism of fertility genes on plants and has great application and promotion value for rice breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of plant phenotype, anther dehiscence, anther morphology, pollen potassium iodide staining and anther semi-thin sections of wild-type Huanghuazhan (HHZ) and EMS mutant oscyp86a9.

[0019] Figure 2 Schematic diagram of SIMM analysis of the OsCYP86A9 gene and the mutation sites of the oscyp86a9 and CR-1 mutants.

[0020] Figure 3 Schematic diagram of the phenotypes of OsCYP86A9 gene knockout and genomic complementation plants.

[0021] Figure 4Semi-thin sections and transmission electron microscopy of anther cross sections of wild-type Huanghuazhan (HHZ) and mutant oscyp86a9. DMs, aborted microspores; E, epidermis; En, endodermis; M, middle layer; MP, mature pollen grain; Ms, microspore; T, tapetum; DP, aborted pollen grain; Ub, ubobiont; Ex, pollen exine; cw, cell wall; c, cuticle. DETAILED DESCRIPTION

[0022] 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.

[0023] Example 1: Obtaining oscyp86a9 mutants

[0024] A mutant with reduced fertility was obtained from the EMS-induced mutant library of Huang Huazhan, which had no significant difference in vegetative growth from the wild type ( Figure 1 A), but the spikelet setting rate decreased significantly ( Figure 1 B). In the mutant, the anthers of some florets can dehisce and release pollen normally, but the anthers of most florets cannot dehisce normally ( Figure 1 C and 1D). Before flowering, a small number of anthers are similar to the wild type, while most anthers are thinner and lighter in color than the wild type ( Figure 1 E and 1F); I2-KI staining showed that pollen in different florets of the mutant was completely dyeable, partially dyeable, and completely abortive ( Figure 1 G and 1H). The semi-thin sections at the 12th stage of anther development showed that different anthers of the mutant had different degrees of pollen abortion, and the degree of pollen abortion in different anther cells of the same anther was also different ( Figure 1 I and 1J).

[0025] Example 2: Localization of OsCYP86A9 gene

[0026] The mutant was hybridized with the wild-type Huanghuazhan, and the F1 generation had normal fertility. In the F2 generation plants, the segregation ratio of plants with normal fertility and plants with reduced fertility was 3:1 (df=1, P=0.05, χ2=3.84), which indicated that the mutant phenotype was caused by a single gene recessive mutation.

[0027] Thirty mutants with reduced fertility were selected from the F2 population constructed by hybridization between mutants and wild-type Huanghuazhan. Genomic DNA was extracted from their leaves and mixed in equal amounts, and then high-throughput sequencing was performed using the Illumina Hiseq 2000 platform. A total of about 13Gb of genome sequencing data was obtained, equivalent to 19× rice genomes. The mutant sequencing data obtained was compared with the Nipponbare reference genome, with an alignment rate of 87.33% and a coverage rate of 85.93%. A total of 1,281,194 SNPs were obtained. By eliminating sites with inconsistent non-mutation ratios and sequencing depths in the control material, candidate intervals for the mutant were obtained. By calculating the mutation frequency and the Euclidean distance ED of the mutation site, the candidate interval of the mutant was screened, and combined with the effect of SNP on the gene, the mutant gene was located on the LOC_Os01g63540 gene on chromosome 1. This gene encodes the OsCYP86A9 protein. The mutant has a single base substitution in the exon (G replaced by A), resulting in a change in amino acids, from Arg 314 (CGC) becomes His 314 (CAC)( Figure 2 ).

[0028] Example 3: CRISPR / Cas9 creates oscyp86a9 allele mutants

[0029] To verify that the OsCYP86A9 mutation can cause rice pollen abortion, the CRISPR / Cas9 system was used to edit the OsCYP86A9 gene. Two specific CRISPR knockout target sites (the 3' end 3 bases are PAM sequences) were designed for the target gene on the CRISPR-GE website (http: / / skl.scau.edu.cn / ). The sequences are as follows: 5'-TCACATGACGGAGACCATCG-3' and 5'-CCCGCGCGTCTCCCTGAGAA-3'.

[0030] 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 was added with a sequence complementary to the sticky end of the backbone vector as the primer of the target, and then the pYLCRISPR / Cas9-MH and gRNA plasmids were treated with BsaⅠ endonuclease to verify whether the plasmid was normal, and then the designed knockout primer was used to make the target linker; 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.

[0031] Molecular identification and sequencing analysis of these transgenic seedlings revealed that the OsCYP86A9 knockout mutant plant CR-1 was obtained. The mutant had a missing base CA in target site 1 and an inserted base A in target site 2 ( Figure 2 ), resulting in a protein frameshift mutation. The mutant plant phenotype is similar to that of the oscyp86a9 mutant, with reduced panicle fruit set, some anthers being thinner and lighter in color than the wild type, and pollen I 2 -KI staining results show that some can be stained and some cannot be stained ( Figure 3 G-3M).

[0032] Example 4: Genome complementation to restore oscyp86a9 mutation virulence

[0033] The function of OsCYP86A9 gene was further verified by gene function complementation experiment. Primers were designed to amplify OsCYP86A9 gene, including 2420bp upstream of the start codon and 1107bp downstream of the stop codon, a total of 5165bp (as shown in SEQ ID NO:3). The fragment was connected to the 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 genome complementation vector was named pCAMBIA1300-OsCYP86A9. The plasmid was then transferred into Agrobacterium, and the target fragment was introduced into the callus tissue of the seeds of the oscyp86a9 mutant heterozygous strain through Agrobacterium-mediated. Plants with a genomic background of homozygous mutation of oscyp86a9 were screened from the obtained transgenic positive plants, and the phenotypes of their offspring were examined. The results showed that the fruiting rate of the transgenic positive plants of the oscyp86a9 mutant was significantly higher than that of the mutant, and there was no difference with the wild type ( Figure 3 E and 3F); the anther color and anther morphology of the oscyp86a9 mutant transgenic positive plants were also restored and were similar to those of the wild type ( Figure 3 A and 3B); Pollen I 2 -KI staining is completely dyeable ( Figure 3 C and 3D), indicating that pollen fertility was restored.

[0034] Example 5: Cytological analysis of mutant oscyp86a9

[0035] To further investigate at which stage of anther development the oscyp86a9 mutant began to show defects, we compared semi-thin sections of the wild type and mutant from pollen mother cell formation (stage 6) to pollen maturity (stage 12). The results showed that there was no obvious difference between the mutant and the wild type before the microspores were released from the tetrad (stage 9). Figure 4 A and 4E), at the 10th stage of microspore vacuolization, the microspores of the mutant were more swollen than those of the wild type ( Figure 4 B and 4F). At stage 11, the microspores of the wild type were sickle-shaped, with dark-stained tapetum, while the microspores of the mutant were deformed, with light-stained and swollen tapetum ( Figure 4 C and 4G). At the 12th period, the pollen grains of the wild type were full, dark in color, and had degraded tapetum, while the pollen grains of the mutant were shrunken, without inclusions, and had abnormal tapetum degradation ( Figure 4 D and 4H).

[0036] To understand more defects of the mutant in anthers, we further observed by transmission electron microscopy. The results showed that compared with the wild type, the inner layer of the pollen outer wall of the mutant was not smooth, had fractures, and the Wuerbach bodies were sparser ( Figure 4 L and 4O, 4M and 4P), but no significant difference was found in anther cuticle ( Figure 4 These results suggest that OsCYP86A9 is involved in the formation of anther exine and that this gene has the function of regulating anther development in rice.

Claims

1. A protein OsCYP86A9 that regulates rice anther development, characterized in that: Any of the following (a)-(c): (a) a protein having the amino acid sequence shown in SEQ ID NO: 1; or (b) a protein having a function of regulating rice anther development, wherein the amino acid sequence in (a) is substituted and / or deleted and / or one or more amino acids are added; or (c) A protein having an amino acid sequence homology of more than 80% with any of the amino acids defined in (a) and (b) and having the same function.

2. A gene for regulating rice anther development, characterized in that: Any of the following (a)-(d): (a) a nucleotide sequence encoding the protein OsCYP86A9 for regulating rice anther development according to claim 1; or (b) a nucleotide sequence that hybridizes with the DNA molecule defined in (a) under stringent conditions and encodes the protein OsCYP86A9 for regulating rice anther development according to claim 1; or (c) having the nucleotide sequence shown in SEQ ID NO: 2 or 3; or (d) A gene having 70% or more homology with any of the nucleotide sequences defined in (a) to (c) and encoding a protein having the same function.

3. A method for causing partial abortion of rice pollen and a decrease in seed setting rate, characterized in that: The method is produced by mutating the OsCYP86A9 protein, wherein the amino acid sequence of the OsCYP86A9 protein is as described in claim 1.

4. A method for obtaining a rice anther development abnormal mutant, characterized in that: include: Using genetic engineering to knock out OsCYP86A9 Gene, wherein the OsCYP86A9 The gene is as described in claim 2.

5. The method according to claim 4, characterized in that Gene OsCYP86A9 The target is a sgRNA sequence based on CRISPR / Cas9, a DNA fragment containing the sgRNA sequence is connected to a vector carrying CRISPR / Cas, and rice is transformed to obtain a mutant with loss of gene function.

6. The method according to claim 4, characterized in that The nucleotide sequences of the sgRNA action site are 5′-TCACATGACGGAGACCATCG-3′ and 5′-CCCGCGCGTCTCCCTGAGAA-3′.

7. A method for restoring the abnormal phenotype of rice anther development, characterized in that: include: Will OsCYP86A9 The promoter and gene region sequence of were introduced into the mutant by genetic engineering; OsCYP86A9 The gene as claimed in claim 2.

8. The method according to claim 7, characterized in that Gene OsCYP86A9 The promoter and gene region sequence of the gene were constructed into a plant binary expression vector and transformed into rice to obtain transgenic plants that restored the mutant phenotype of the gene.

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