Application of rice endogenous gene combination for regulating and controlling important agronomic traits and cuticle development in breeding

Abstract

The invention provides an application of a rice endogenous gene combination for regulating and controlling important agronomic traits and stratum corneum development in breeding, and particularly relates to two highly similar genes OsCYP86A10 and OsCYP86A11. Plant phenotypes, such as plant height, ear length, flag leaf length, grain length, thousand seed weight, effective division number, permeability of leaf cuticle and the like, can be regulated and controlled by using the two proteins or coding genes independently or in a combined manner, and a foundation is laid for improving important agricultural traits of rice.

Description

[0001] field of study

[0002] The present invention relates to the field of plant genetic engineering, and in particular to the application of a combination of endogenous rice genes for regulating important agronomic traits and cuticle development in breeding.

[0003] Background

[0004] In higher plants, the cuticle covers the outer epidermal surface of most above-ground tissues, such as leaves, fruits, and floral organs. The cuticle is best known for its function as a diffusion barrier, limiting the transport of water and solutes in the apoplast and protecting the plant from chemical and mechanical damage and attack by pests and pathogens.

[0005] The cuticle is mainly composed of cutin and wax. The main chemical components of cutin are C16 and C18 fatty acids and propylene glycol, and wax is usually composed of C20 to C36 very long chain saturated fatty acids, including alkanes, aldehydes, ketones, primary alcohols and secondary alcohols. The structure of the cuticle has relatively clear stratification, but the thickness and structure of each layer vary to varying degrees in different species and plant tissues and organs. The structure of the cuticle can usually be divided into three layers. Among them, the cuticle base is rich in cutin and is a cuticle embedded with cell wall polysaccharides; covering the cuticle base is the true cuticle, which is rich in wax and has less cell wall polysaccharides; the outermost cuticle usually has only wax components, and the wax on its surface is often deposited as a layer of wax film and / or wax crystals.

[0006] In the cytochrome P450 superfamily, the genes related to the cuticle that have been reported so far include CYP703A3, CYP704B2, CYP77A6, CYP86A2, CYP86A4, and CYP86A8. The protein encoded by CYP703A3 is a fatty acid hydroxylase, which is specifically expressed in the tapetum and is essential for rice male fertility. The cyp703a3-2 mutant exhibits defects in the pollen exine and anther cuticle. Chemical analysis shows that the content of anther cutin monomers and wax components in the cyp703a3-2 mutant is significantly reduced. CYP704B2 is expressed in the tapetum and microspores from the 8th to the 10th stage of anther development, catalyzing the ω-position hydroxylation of carbon chains with 16 and 18 carbons. Chemical composition analysis found that the cutin monomers of the mutant anthers were reduced. This indicates that CYP704B2 is involved in the biosynthesis of anther cutin. CYP77A6 is highly expressed in the petals of Arabidopsis thaliana. After mutation, the dihydroxy cutin monomers on the petals are completely lost. Ultrastructural observation shows that the true cuticle and ultrastructure ridges on the petals of the mutant are missing. The CYP77A6 protein has been shown to catalyze the hydroxylation of the middle of the carbon chain and participate in the formation of the cutin monomer 10,16-dihydroxyhexadecanoic acid. The hydroxylated fatty acids will be transferred to glycerol-3-phosphate (GPATs) by glycerol-3-phosphate acyltransferase, which is an enzyme involved in cutin formation. ATT1 encodes CYP86A2, a cytochrome P450 monooxygenase, which catalyzes the oxidation of fatty acids. The att1 cutin monomer content is reduced, the cuticle membrane structure is loose, and the permeability to water is increased. After CYP86A4 mutation, its cutin composition changes. LCR encodes the cytochrome P450 monooxygenase protein CYP86A8, which catalyzes the ω hydroxylation of fatty acids from C12 to C18:1, and its effective substrates are oleic acid and palmitic acid. LCR mutations manifest various developmental abnormalities, including abnormalities in the structure of the cuticle and fusion of reproductive organs.

[0007] The two genes OsCYP86A10 and OsCYP86A11 in rice have not been reported yet. They are homologous genes to the CYP86A2, CYP86A2 and CYP86A8 genes of Arabidopsis. The gene functions of OsCYP86A10 and OsCYP86A11 are also related to the cuticle. The cuticle plays a vital role in drought resistance. Analyzing the functions of OsCYP86A10 and OsCYP86A11 in the formation of plant cuticle can not only improve the regulatory network of plant cuticle formation, but also provide a molecular mechanism as a reference for the breeding of drought-resistant rice varieties. Summary of the invention

[0008] One object of the present invention is to provide two genes OsCYP86A10 and OsCYP86A11 and their encoded proteins that regulate important agronomic traits of rice (such as plant height, effective tiller number, panicle length, flag leaf length, grain length, grain width or 1000-grain weight, etc.) and cuticle development.

[0009] The nucleotide sequences of genes OsCYP86A10 and OsCYP86A11 are shown in SEQ ID NO: 2 or 4, respectively, and the sequences of proteins OsCYP86A10 and OsCYP86A11 encoded by them are shown in SEQ ID NO: 1 and 3, respectively.

[0010] Another object of the present invention is to provide a method for causing rice plants to produce a dwarf phenotype or abnormal cuticle development. OsCYP86A10 and OsCYP86A11 were knocked out by gene editing, and functional loss mutants oscyp86a10 and oscyp86a11 were obtained. Among them, the oscyp86a10 mutant lacks a guanine (G) at the 493rd base after ATG, resulting in a frameshift mutation in the protein; the oscyp86a11 mutant lacks 6 bases CCTCAA after the 1124th base after ATG, resulting in the formation of the terminator TAA at the mutant sequence, thereby terminating the protein prematurely.

[0011] The two genes OsCYP86A10 and OsCYP86A11 described in the present invention encode proteins of the amino acid sequences shown in SEQ ID NO: 1 and 3 in the sequence list, respectively. The amino acid sequence of SEQ ID NO: 1 in the sequence list consists of 533 amino acid residues, and the protein of the oscyp86a10 mutant undergoes a frameshift mutation after the 164th amino acid; the amino acid sequence of SEQ ID NO: 3 in the sequence list consists of 546 amino acid residues, and the protein of the oscyp86a11 mutant terminates at the 375th amino acid, resulting in premature termination of the protein.

[0012] The gene encoding the above protein may be a cDNA sequence of the gene, such as the nucleotide sequence shown in SEQ ID NO: 2 and SEQ ID NO: 4 in the sequence table. In the oscyp86a10 mutant, the 493rd base guanine (G) is missing; in the oscyp86a11 mutant, 6 bases CCTCAA are missing after the 1124th base.

[0013] Another object of the present invention is to provide a method for obtaining a rice plant with a dwarf phenotype or a mutant with abnormal cuticle development. OsCYP86A10 and OsCYP86A11 were knocked out by gene editing, and the functional mutants oscyp86a10 and oscyp86a11 were obtained. Among them, the oscyp86a10 mutant lacks a guanine (G) at the 493rd base after ATG, resulting in a frameshift mutation in the protein. Its nucleic acid sequence is shown in the sequence list SEQ ID NO: 6, and its protein sequence is shown in the sequence list SEQ ID NO: 5; the oscyp86a11 mutant lacks 6 bases CCTCAA after the 1124th base after ATG, resulting in the formation of a terminator TAA in the mutant sequence, thereby causing the protein to terminate prematurely. Its nucleic acid sequence is shown in the sequence list SEQ ID NO: 8, and its protein sequence is shown in the sequence list SEQ ID NO: 7.

[0014] The two genes OsCYP86A10 and OsCYP86A11 described in the present invention encode proteins of the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3 in the sequence list, respectively. The amino acid sequence of SEQ ID NO: 1 in the sequence list consists of 533 amino acid residues, and the protein of the oscyp86a10 mutant undergoes a frameshift mutation after the 164th amino acid, and the protein sequence is shown in SEQ ID NO: 5 in the sequence list; the amino acid sequence of SEQ ID NO: 3 in the sequence list consists of 546 amino acid residues, and the protein of the oscyp86a11 mutant terminates at the 375th amino acid, resulting in premature termination of the protein, and the protein sequence is shown in SEQ ID NO: 7 in the sequence list.

[0015] The gene encoding the above protein may be a cDNA sequence of the gene, such as the nucleotide sequence shown in SEQ ID NO: 2 and SEQ ID NO: 4 in the sequence table. In the oscyp86a10 mutant, the 493rd base guanine (G) is missing; in the oscyp86a11 mutant, 6 bases CCTCAA are missing after the 1124th base.

[0016] The present invention finds that the plant height, ear length, sword leaf length, grain length and 1000-grain weight of the oscyp86a10 mutant plant are significantly reduced compared with the wild type; the oscyp86a11 mutant has no effect on the macroscopic phenotype of the plant; when OsCYP86A10 is a homozygous mutation and OsCYP86A11 is a heterozygous mutation, the plant height, effective tiller number, ear length, sword leaf length, grain length, grain width and 1000-grain weight of the plant are more severely inhibited than those of the oscyp86a10 mutant; when OsCYP86A10 is a homozygous mutation and OsCYP86A11 is also a homozygous mutation, the plant is short and difficult to survive.

[0017] The two rice genes described in the present invention affect the permeability of the rice cuticle after mutation: when OsCYP86A10 is a homozygous mutation and OsCYP86A11 is a heterozygous mutation, compared with the wild type and two single mutants of oscyp86a10 and oscyp86a11, their leaves have a faster water loss rate, darker toluidine blue staining and faster chlorophyll leaching speed.

[0018] The plant phenotype-related proteins OsCYP86A10 and OsCYP86A11 and their encoding genes provided by the present invention can regulate plant height, panicle length, flag leaf length, grain length, and 1000-grain weight, and other phenotypes, which are of great significance to rice molecular breeding. After the mutation of OsCYP86A10 and OsCYP86A11, the accumulation of leaf cuticle is affected, providing a reference for the later breeding of new drought-resistant rice varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of phenotypic analysis of wild type, oscyp86a10, oscyp86a11 and oscyp86a10 oscyp86a11 (+ / -) mutants.

[0020] Figure 2 Figures 2A and 2B show the permeability test of leaves of wild type, oscyp86a10, oscyp86a11 and oscyp86a10 oscyp86a11(+ / -) mutants. Figures A, B and C show the water loss rate test, toluidine blue staining and chlorophyll leaching experiment of leaves, respectively. DETAILED DESCRIPTION

[0021] 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.

[0022] Example 1: Gene editing CRISPR / Cas9 to create oscyp86a10 and oscyp86a11 mutants

[0023] OsCYP86A10 (amino acid sequence as shown in SEQ ID NO: 1, nucleotide sequence as shown in SEQ ID NO: 2) and OsCYP86A11 (amino acid sequence as shown in SEQ ID NO: 3, nucleotide sequence as shown in SEQ ID NO: 4) are two very similar genes, with the nucleotide sequence similarity of the coding region reaching 86.4% and the protein sequence similarity reaching 87.7%. To explore the gene functions of OsCYP86A10 and OsCYP86A11, gene editing of OsCYP86A10 and OsCYP86A11 was performed by CRISPR / Cas9. A specific CRISPR knockout target site (the 3′ end 3 bases are PAM sequence) was designed for each target gene on the CRISPR-GE website (http: / / skl.scau.edu.cn / ), where the sequence of OsCYP86A10 is as follows: 5′-GCTGCCCATCCTGGACGACG-3′, and the sequence of OsCYP86A11 is as follows: 5′-CCAGCTCGTCTACCTCAAAG-3′.

[0024] 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.

[0025] These transgenic seedlings were molecularly identified and sequenced to obtain the corresponding mutants oscyp86a10 and oscyp86a11. The mutation of oscyp86a10 occurred at 493bp after ATG, with a missing base G (as shown in SEQ ID NO:6), resulting in a protein frameshift mutation (as shown in SEQ ID NO:5). The mutation of oscyp86a11 occurred 1124bp after ATG, with a missing 6 bases CCTCAA (as shown in SEQ ID NO:8), resulting in the formation of the terminator TAA at this location in the mutant sequence, causing the protein to terminate prematurely (as shown in SEQ ID NO:7).

[0026] Example 2: Phenotypic analysis of oscyp86a10 and oscyp86a11 mutants

[0027] The plants mutated by OsCYP86A10 became shorter, with shorter panicle, flag leaf and grain lengths and lighter thousand-grain weights, but the number of effective divisions and number of grains per panicle were not significantly different from those of the wild type ( Figure 1 A, 1C, 1E, 1F and 1H). The OsCYP86A11 mutation had no effect on the growth and development of the plant ( Figure 1 A, 1C, 1E, 1F and 1H). When the genotype of OsCYP86A10 is a homozygous mutation and the genotype of OsCYP86A11 is a heterozygous mutation, the height of the plants with this genotype is shorter than that of the plants with OsCYP86A10 mutation alone, the number of effective divisions is reduced, the panicle length, flag leaf length and grain length are shortened, the grain width is narrowed, the seeds are smaller, the 1000-grain weight is lighter, and the husk is brown ( Figure 1 A, 1C, 1E, 1F and 1H), the chlorophyll content of the sword leaf at the mature stage was higher than that of the plants of other genotypes ( Figure 1 D) The endosperm of oscyp86a10oscyp86a11(+ / -) seeds was greener compared with the endosperm of wild-type, OsCYP86A10 and OsCYP86A11 seeds at the same time. Figure 1 G), matures relatively slowly. When both OsCYP86A10 and OsCYP86A11 genotypes are homozygous mutants, the plants are short and difficult to survive ( Figure 1 C). The above results indicate that in the case of OsCYP86A10 mutation, OsCYP86A11 has a dosage effect; in regulating plant growth and development, OsCYP86A10 and OsCYP86A11 play an important role and there is gene functional redundancy. Compared with OsCYP86A11, OsCYP86A10 plays a major role.

[0028] Example 3: Mutations in OsCYP86A10 and OsCYP86A11 affect stratum corneum permeability

[0029] The leaf water loss rates of the wild type, oscyp86a10, oscyp86a11, and oscyp86a10 oscyp86a11(+ / -) mutants were compared. The results showed that the oscyp86a10 oscyp86a11(+ / -) mutant had the fastest water loss rate, suggesting that the mutant had a defect in its cuticle.

[0030] Toluidine blue is a dye that can detect whether there are defects in the cuticle or epidermis. The leaves of the wild type and oscyp86a10, oscyp86a11, and oscyp86a10 oscyp86a11(+ / -) were stained with 0.5% toluidine blue solution. The results showed that the leaves of the oscyp86a10 oscyp86a11(+ / -) mutant were stained significantly darker than those of the other genotypes (Figure 4B), suggesting that the cuticle permeability of the mutant was increased.

[0031] Using 80% ethanol to detect the leaching of chlorophyll from leaves is also a method to determine the permeability of the cuticle. The leaves of the wild type and the oscyp86a10 oscyp86a11 (+ / -) mutant were selected and placed in 80% ethanol for chlorophyll extraction. The results showed that the leaching rate of chlorophyll from the leaves of the oscyp86a10oscyp86a11 (+ / -) mutant was faster than that of the wild type (Figure 4C), indicating that the permeability of the cuticle of the oscyp86a10oscyp86a11 (+ / -) mutant was enhanced. All of these results indicate that the mutation of OsCYP86A10 and OsCYP86A11 affects the permeability of the cuticle, and further suggest that these two genes are related to the formation of the cuticle.

Claims

1. A method for causing a rice plant to have a dwarf phenotype or abnormal cuticle development, characterized in that: By simultaneously mutating proteins OsCYP86A10 and OsCYP86A11, wherein the amino acid sequences of the proteins OsCYP86A10 and OsCYP86A11 are shown in SEQ ID NOs: 1 and 3, respectively.

2. The method of claim 1, wherein the gene sequence encoding the protein OsCYP86A10 is shown in SEQ ID NO: 2, and the gene sequence encoding the protein OsCYP86A11 is shown in SEQ ID NO:

4.

3. The method of claim 1, wherein the simultaneous mutation of proteins OsCYP86A10 and OsCYP86A11 is performed by genetic engineering to simultaneously knock out the OsCYP86A10 and OsCYP86A11 Genes, among which The gene sequence is as described in claim 2.

4. The method according to claim 3, characterized in that: Genetic engineering is the process of OsCYP86A10 and OsCYP86A11 The two genes are targeted and a sgRNA sequence based on CRISPR / Cas9 is designed. The DNA fragment containing the sgRNA sequence is connected to a vector carrying CRISPR / Cas9 and transformed into rice to obtain transgenic rice with the two genes missing.

5. The method according to claim 4, characterized in that: OsCYP86A10 The nucleotide sequence of the sgRNA action site of the gene is 5'-GCTGCCCATCCTGGACGACG-3', OsCYP86A11 The nucleotide sequence of the sg RNA action site of the gene is 5'-CCAGCTCGTCTACCTCAAAG-3'.

6. A method for obtaining a rice plant with a dwarf phenotype or a mutant with abnormal cuticle development, characterized in that: By simultaneously mutating proteins OsCYP86A10 and OsCYP86A11, wherein the amino acid sequences of the proteins OsCYP86A10 and OsCYP86A11 are shown in SEQ ID NOs: 1 and 3, respectively.

7. The method of claim 6, wherein the gene sequence encoding the protein OsCYP86A10 is shown in SEQ ID NO: 2, and the gene sequence encoding the protein OsCYP86A11 is shown in SEQ ID NO:

4.

8. The method of claim 6, wherein the simultaneous mutation of proteins OsCYP86A10 and OsCYP86A11 is performed by genetic engineering to simultaneously knock out the OsCYP86A10 and OsCYP86A11 Genes, among which The gene sequence is as described in claim 7.

9. The method according to claim 8, characterized in that: Genetic engineering is the process of OsCYP86A10 and OsCYP86A11 The two genes are targeted and a sgRNA sequence based on CRISPR / Cas9 is designed. The DNA fragment containing the sgRNA sequence is connected to a vector carrying CRISPR / Cas9 and transformed into rice to obtain transgenic rice with the two genes missing.

10. The method according to claim 9, characterized in that: OsCYP86A10 The nucleotide sequence of the sgRNA action site of the gene is 5'-GCTGCCCATCCTGGACGACG-3', OsCYP86A11 The nucleotide sequence of the sg RNA action site of the gene is 5'-CCAGCTCTACCTCAAAG-3'.

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