Application of a combination of endogenous rice genes regulating important agronomic traits and cuticle development in breeding

Abstract

The application provides application of a combination of endogenous rice genes in regulating important agronomic traits and cuticle development in breeding, and particularly relates to two highly similar genes OsCYP86A10 and OsCYP86A11 . The two proteins or coding genes are used alone or in combination to regulate plant phenotypes, such as plant height, ear length, flag leaf length, grain length, thousand-grain weight, effective spikelet number, and leaf cuticle permeability, thereby laying a foundation for improving important agricultural traits of rice.

Description

[0001] field of study

[0002] This invention relates to the field of plant genetic engineering, specifically to the application of a combination of endogenous rice genes that regulate important agronomic traits and cuticle development in breeding.

[0003] Research Background

[0004] In higher plants, the cuticle covers the outer surface of the epidermis of most terrestrial tissues, such as leaves, fruits, and floral organs. The cuticle is best known for its function as a diffusion barrier, restricting the transport of water and solutes in the apoplast and protecting plants from chemical and mechanical damage as well as attacks by pests and pathogens.

[0005] The cuticle is mainly composed of cuticle and wax. The main chemical components of cuticle are C16 and C18 fatty acids and glycerol, while wax is usually composed of C20 to C36 ultra-long-chain saturated fatty acids, including alkanes, aldehydes, ketones, primary alcohols, and secondary alcohols. The cuticle has a relatively clear layered structure, but the thickness and structure of each layer vary to varying degrees in different species and plant tissues and organs. The cuticle can generally be divided into three layers: the basal layer, rich in cuticle, is a cuticle layer embedded with cell wall polysaccharides; covering the basal layer is the true cuticle, which is rich in wax and has fewer cell wall polysaccharides; the outermost cuticle usually consists only of wax, and its surface wax is often deposited as a waxy film and / or wax crystals.

[0006] Within the cytochrome P450 superfamily, genes reported to be related to the cuticle include CYP703A3, CYP704B2, CYP77A6, CYP86A2, CYP86A4, and CYP86A8. CYP703A3 encodes a fatty acid hydroxylase, which is specifically expressed in the tapetum and is crucial for male fertility in rice. The cyp703a3-2 mutant exhibits defects in the pollen exine and anther cuticle. Chemical analysis shows a significant decrease in the content of anther cuticle monomers and waxy components in the cyp703a3-2 mutant. CYP704B2 is expressed in the tapetum and microspores during anther development stages 8 to 10, catalyzing the ω-position hydroxylation of 16-carbon and 18-carbon carbon chains. Chemical composition analysis revealed a reduction in cuticle monomers in the mutant anthers. This indicates that CYP704B2 is involved in the biosynthesis of anther cuticle. CYP77A6 is highly expressed in the petals of Arabidopsis thaliana. Mutants in this variant completely lose the dihydroxykeratin monomer in the petals, and ultrastructural observation reveals the absence of the eukeratin layer and ultra-ridged structures on the petals. The CYP77A6 protein has been shown to catalyze hydroxylation in the middle of the carbon chain, participating in the formation of the keratin monomer 10,16-dihydroxyhexadecanoic acid. The hydroxylated fatty acids are then transferred to glycerol-3-phosphate (GPATs) by glycerol-3-phosphoacyltransferases, enzymes involved in keratin formation. ATT1 encodes CYP86A2, a cytochrome P450 monooxidase that catalyzes fatty acid oxidation. ATT1 reduces the content of keratin monomers, resulting in a looser cuticle membrane structure and increased water permeability. Mutations in CYP86A4 alter the composition of the keratin. LCR encodes the cytochrome P450 monooxidase protein CYP86A8, catalyzing the ω-hydroxylation of fatty acids from C12 to C18:1, with oleic acid and palmitic acid as effective substrates. LCR mutations manifest as various developmental abnormalities, including abnormal cuticle structure and fusion of reproductive organs.

[0007] The genes OsCYP86A10 and OsCYP86A11 in rice have not yet been reported. They are homologous to the CYP86A2, CYP86A2, and CYP86A8 genes in Arabidopsis thaliana. The functions of OsCYP86A10 and OsCYP86A11 are also related to the cuticle, which plays a crucial role in drought resistance. Elucidating the functions of OsCYP86A10 and OsCYP86A11 in the process of plant cuticle formation can not only improve the regulatory network of plant cuticle formation, but also provide a molecular mechanism 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, number of effective tillers, panicle length, flag leaf length, grain length, grain width or thousand-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 the proteins they encode, OsCYP86A10 and OsCYP86A11, are shown in SEQ ID NO:1 and 3, respectively.

[0010] Another objective of this invention is to provide a method for inducing dwarfism or abnormal cuticle development in rice plants. By performing site-specific knockout of OsCYP86A10 and OsCYP86A11 through gene editing, loss-of-function mutants oscyp86a10 and oscyp86a11 were obtained. Specifically, the oscyp86a10 mutant involves the deletion of a guanine (G) nucleotide at position 493 after the ATG, resulting in a frameshift mutation in the protein; the oscyp86a11 mutant involves the deletion of six CCTCAA nucleotides after position 1124 after the ATG, causing the mutant sequence to form a terminator TAA at this position, thus prematurely terminating the protein.

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

[0012] The gene encoding the above-mentioned protein can be the cDNA sequence of the gene, such as the nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 4 in the sequence listing. In the oscyp86a10 mutant, guanine (G) is deleted at base 493; in the oscyp86a11 mutant, six bases CCTCAA are deleted after base 1124.

[0013] Another objective of this invention is to provide a method for obtaining rice plants with a dwarf phenotype or abnormal cuticle development mutants. By gene editing, OsCYP86A10 and OsCYP86A11 were knocked out at specific sites, resulting in loss-of-function mutants oscyp86a10 and oscyp86a11. Specifically, the oscyp86a10 mutant has a deletion of a guanine (G) at position 493 after the ATG, leading to a frameshift mutation in the protein. Its nucleic acid sequence is shown in SEQ ID NO: 6, and its protein sequence is shown in SEQ ID NO: 5. The oscyp86a11 mutant has a deletion of 6 bases CCTCAA after position 1124 after the ATG, resulting in the formation of a terminator TAA at this position, causing premature protein termination. Its nucleic acid sequence is shown in SEQ ID NO: 8, and its protein sequence is shown in SEQ ID NO: 7.

[0014] The two genes OsCYP86A10 and OsCYP86A11 described in this invention encode proteins with the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3, respectively, in the sequence listing. The amino acid sequence of SEQ ID NO: 1 consists of 533 amino acid residues, and the protein of its oscyp86a10 mutant has a frameshift mutation after amino acid position 164, as shown in SEQ ID NO: 5. The amino acid sequence of SEQ ID NO: 3 consists of 546 amino acid residues, and the protein of its oscyp86a11 mutant terminates at amino acid position 375, resulting in premature protein termination, as shown in SEQ ID NO: 7.

[0015] The gene encoding the above-mentioned protein can be the cDNA sequence of the gene, such as the nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 4 in the sequence listing. In the oscyp86a10 mutant, guanine (G) is deleted at base 493; in the oscyp86a11 mutant, six bases CCTCAA are deleted after base 1124.

[0016] This invention found that the plant height, spike length, flag leaf length, grain length, and thousand-grain weight of the oscyp86a10 mutant were significantly reduced compared to the wild type; the oscyp86a11 mutant had no effect on the macroscopic phenotype of the plant; when OsCYP86A10 was a homozygous mutant and OsCYP86A11 was a heterozygous mutant, the plant height, effective tiller number, spike length, flag leaf length, grain length, grain width, and thousand-grain weight were more severely suppressed compared to the oscyp86a10 mutant; when both OsCYP86A10 and OsCYP86A11 were homozygous mutants, the plants were dwarfed and had difficulty surviving.

[0017] The two rice genes described in this 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 the two single mutants oscyp86a10 and oscyp86a11, the leaves lose water faster, the toluidine blue staining is deeper, and the chlorophyll leaching rate is faster.

[0018] The plant phenotype-related proteins OsCYP86A10 and OsCYP86A11, along with their encoding genes, provided by this invention, can regulate plant height, panicle length, flag leaf length, grain length, and thousand-grain weight, among other phenotypes. These agronomic traits are of great significance for molecular breeding of rice. Mutations in OsCYP86A10 and OsCYP86A11 affect the accumulation of leaf cuticle, providing a reference for the later selection of drought-resistant rice varieties. Attached Figure Description

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

[0020] Figure 2 Permeability was measured in leaves of wild type, oscyp86a10, oscyp86a11, and oscyp86a10 oscyp86a11(+ / -) mutants. Figures A, B, and C show the leaf water loss rate, toluidine blue staining, and chlorophyll leaching experiments, respectively. Detailed Implementation

[0021] The present invention will be further described below with reference to specific embodiments to facilitate better understanding, but this does not limit the scope of the invention.

[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 a nucleotide sequence similarity of 86.4% and a protein sequence similarity of 87.7% in their coding regions. To investigate the gene functions of OsCYP86A10 and OsCYP86A11, gene editing of OsCYP86A10 and OsCYP86A11 was performed using CRISPR / Cas9. A specific CRISPR knockout target site (with the 3' last 3 bases being PAM sequences) was designed for each target gene on the CRISPR-GE website (http: / / skl.scau.edu.cn / ). The OsCYP86A10 sequence is as follows: 5'-GCTGCCCATCCTGGACGACG-3', and the OsCYP86A11 sequence is as follows: 5'-CCAGCTCGTCTACCTCAAAG-3'.

[0024] Gene knockout vectors were constructed using the pYLCRISPR / Cas9-MH vector provided by Professor Liu Yaoguang's research group at South China Agricultural University. The specific construction steps followed the accompanying operating guidelines provided by the research group. Primers for the target vector were added with sequences complementary to the sticky ends of the backbone vector. Primers for the knockout target vector were synthesized by BGI Genomics. Then, pYLCRISPR / Cas9-MH and gRNA plasmids were treated with BsaI restriction enzyme to verify plasmid functionality. Next, target adapters were created using the designed knockout primers. The target adapters and gRNA expression cassettes were then ligated together using a cut-and-ligate method with BsaI restriction enzyme and T4 DNA ligase. Amplification was performed through two rounds of PCR, and the target band was detected by gel electrophoresis. The recovered product was then excised and its concentration was measured. Finally, the recovered target fragment was ligated into the pYLCRISPR / Cas9-MH knockout vector using BsaI restriction enzyme and T4 DNA ligase. The ligation product was heat-transformed into *E. coli*, and positive clones were selected for colony shaking, plasmid extraction, and sequencing. The correctly sequenced plasmid was transferred into Agrobacterium tumefaciens and then introduced into callus tissue through genetic transformation of rice for tissue culture to obtain the corresponding transgenic seedlings.

[0025] Molecular identification and sequencing analysis of these transgenic seedlings yielded the corresponding mutants oscyp86a10 and oscyp86a11. The mutation in oscyp86a10 occurs 493 bp after the ATG, deleting the base G (as shown in SEQ ID NO:6), resulting in a frameshift mutation in the protein (as shown in SEQ ID NO:5). The mutation in oscyp86a11 occurs 1124 bp after the ATG, deleting 6 bases CCTCAA (as shown in SEQ ID NO:8), causing the mutant sequence to form the terminator TAA at this location, leading to premature protein termination (as shown in SEQ ID NO:7).

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

[0027] The OsCYP86A10 mutation resulted in shorter plants, shorter spikelets, flag leaves, and grains, as well as a lighter thousand-grain weight, but the number of effective divisions and the number of grains per spike were not significantly different from the wild type. Figure 1 A, 1C, 1E, 1F, and 1H). The OsCYP86A11 mutation had no effect on plant growth and development. Figure 1 A, 1C, 1E, 1F, and 1H). When the genotype of OsCYP86A10 is homozygous and the genotype of OsCYP86A11 is heterozygous, the plants with this genotype are shorter than those with the OsCYP86A10 monomutation, have fewer effective cell divisions, shorter spike length, flag leaf length, and grain length, narrower grain width, smaller seeds, lighter thousand-grain weight, and brown glumes. Figure 1 A, 1C, 1E, 1F, and 1H) have higher chlorophyll content in their sword-shaped leaves at maturity compared to plants of other genotypes. Figure 1 D), compared with the endosperm of wild-type seeds and single-mutant seeds of OsCYP86A10 and OsCYP86A11 at the same time, the endosperm of OsCYP86A10 and OsCYP86A11 (+ / -) seeds was greener. Figure 1 G), maturation is relatively slower. When both OsCYP86A10 and OsCYP86A11 genotypes are homozygous mutants, the plants are stunted and have difficulty surviving. Figure 1 B). The above results indicate that OsCYP86A11 exhibits a dose-response effect under the OsCYP86A10 mutation; both OsCYP86A10 and OsCYP86A11 play important roles in regulating plant growth and development and exhibit gene functional redundancy, with OsCYP86A10 playing a major role compared to OsCYP86A11.

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

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

[0030] Toluidine blue is a dye that can detect defects in the cuticle or epidermis. A 0.5% toluidine blue solution was used to stain leaves of wild-type and oscyp86a10, oscyp86a11, and oscyp86a10 oscyp86a11(+ / -). The results showed that the leaves of the oscyp86a10 oscyp86a11(+ / -) mutant were stained significantly darker than those of other genotypes. Figure 2 B) This suggests that the mutant has increased keratinocyte permeability.

[0031] Using 80% ethanol to detect chlorophyll leaching from leaves is also a method to assess cuticle permeability. Leaves of wild-type and oscyp86a10 / oscyp86a11(+ / -) mutants were subjected to chlorophyll extraction in 80% ethanol. The results showed that the chlorophyll leaching rate of the oscyp86a10 / oscyp86a11(+ / -) mutant leaves was faster than that of the wild-type. Figure 2 C) indicates that the oscyp86a10 and oscyp86a11 (+ / -) mutants have enhanced stratum corneum permeability. These results demonstrate that mutations in OsCYP86A10 and OsCYP86A11 affect stratum corneum permeability, further suggesting that these two genes are associated with stratum corneum formation.

Claims

1. A method for inducing a dwarf phenotype in rice plants, characterized in that, The OsCYP86A10 gene was knocked out using genetic engineering to obtain a homozygous mutant of OsCYP86A10, the amino acid sequence of which is shown in SEQ ID NO:

1.

2. The method according to claim 1, wherein the coding gene sequence of OsCYP86A10 is shown in SEQ ID NO:

2.

3. The method according to claim 2, wherein the genetic engineering means is to use gene editing methods to perform site-specific knockout of the OsCYP86A10 gene in rice.

4. The method according to claim 3, characterized in that, The genetic engineering method involves using the gene OsCYP86A10 as a target, designing a CRISPR / Cas9-based sgRNA sequence, ligating a DNA fragment encoding the sgRNA sequence into a vector carrying CRISPR / Cas, transforming rice, and obtaining transgenic rice with loss of function.

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

6. A method for inducing dwarfism or abnormal cuticle development in rice plants, characterized in that, By simultaneously knocking out the OsCYP86A10 and OsCYP86A11 genes through genetic engineering, a mutant with homozygous OsCYP86A10 and heterozygous OsCYP86A11 is obtained, wherein the amino acid sequences of OsCYP86A10 and OsCYP86A11 are shown in SEQ ID NO: 1 and 3, respectively.

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

4.

8. The method according to claim 7, wherein the genetic engineering means is to use gene editing methods to perform site-specific knockout of the OsCYP86A10 and OsCYP86A11 genes in rice.

9. The method according to claim 8, characterized in that, The genetic engineering method involves using the genes OsCYP86A10 or OsCYP86A11 as targets, designing sgRNA sequences based on CRISPR / Cas9, ligating DNA fragments encoding the sgRNA sequences into vectors carrying CRISPR / Cas, transforming rice, and obtaining transgenic rice with loss of function.

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

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