A rice gene OsRAL5 that coordinately regulates yield and quality

Abstract

The present invention provides a rice gene OsRAL5 for synergistically regulating yield and quality, and relates to the field of gene engineering technology. The nucleotide sequence of the rice gene OsRAL5 is shown in SEQ ID NO.1, and the protein sequence of the rice gene OsRAL5 is shown in SEQ ID NO.2. The present invention discloses for the first time that the rice OsRAL5 gene has the biological function of synergistically regulating rice yield and quality. It is confirmed that the functional loss and overexpression of the rice OsRAL5 gene can affect the expression of genes related to seed storage proteins and starch synthesis, and the gene expression level is consistent with the change in seed storage protein content. It is further found that the rice OsRAL5 gene has a certain complementary effect on gene expression with homologous genes OsRAG1, OsRAG2, OsRAL2, and OsRAL6.

Description

Technical Field

[0001] The invention relates to the technical field of gene engineering, and in particular to a rice gene OsRAL5 for synergistically regulating yield and quality. Background Art

[0002] Grain size is one of the important factors affecting rice yield. The main factors affecting rice yield are the number of effective panicles, the number of panicles, the fruit set rate and the thousand-grain weight. The thousand-grain weight is mainly determined by the grain size and the fullness, and the grain size is jointly determined by the grain length, grain width, grain thickness and grain fullness. These traits are the factors with the highest heritability among the factors determining rice yield. At the same time, the quality of rice mainly depends on the content of seed storage substances - starch, protein, fat, vitamins and other trace elements. The storage protein content is one of the main factors determining the nutritional quality of rice. Therefore, cloning the genes that control rice yield and quality traits and studying their functions in grain formation and material synthesis accumulation will help to ultimately reveal the molecular regulatory basis of rice yield and quality, which has important theoretical significance and application value for rice genetic improvement.

[0003] According to solubility characteristics, storage proteins can be divided into glutenin, alcohol-soluble protein, albumin and globulin (Yang et al., Genet. Molecul. Biol. 2013, 36, 87–92). Glutelin is the main storage protein of rice, accounting for 60%-80% of seed protein. According to the similarity of amino acid sequence, glutenin is divided into four subfamilies (GluA, GluB, GluC and GluD), with 15 encoding genes (Kawakatsu et al, J. Exp. Bot. 2008, 59, 4233–4245). Alcohol-soluble protein accounts for 20%-30% of seed protein, encoded by a multigene family consisting of 34 gene copies, and can be divided into three subfamilies according to its relative molecular weight (Saito et al, Plant J.: Cell Molecul. Biol. 2012, 70, 1043–1055). Albumin and globulin are water-soluble proteins, accounting for about 5% of seed protein. They belong to the α-amylase / trypsin inhibitor family and show heterogeneity in molecular weight (14-16 kDa) and isoelectric point (pI 6-8). They can specifically bind to immunoglobulin E in allergic people and cause allergic reactions. They are allergen proteins (Tsukasa-Masuda et al, Agric. Biol. Chem. 1991, 55, 509-513).

[0004] The rice OsRAL5 gene encodes a soluble protein in seed storage proteins and belongs to the α-amylase / trypsin inhibitor family. There are 5 homologous genes. So far, the function of this family gene in rice has been less studied (Zhou et al, 2017). By comparing the OsRAL5 protein sequence, it was found that its homologous genes are ubiquitous in plants and are specifically expressed in the endosperm of rice seeds in the late stage of development (Kurokawa et al, Plant Cell Rep. 2014, 33, 75-87), but its biological function in rice seed formation is still unclear. Summary of the invention

[0005] In view of the above-mentioned prior art, the present invention, through gene editing of the sequence shown in SEQ ID NO.1 (OsRAL5 gene) created by transgenic and grain and quality testing of overexpressed rice plants, found that the gene can synergistically positively regulate rice grain size, 1000-grain weight and rice quality. Therefore, the gene has good application value in breeding high-yield and high-quality rice varieties, thereby providing the possibility for breeding new high-yield and high-quality rice varieties.

[0006] The present invention provides a rice gene OsRAL5 for coordinated regulation of yield and quality. The nucleotide sequence of the rice gene OsRAL5 is shown in SEQ ID NO.1, which is 537 bp in total.

[0007] The present invention also provides a rice α-amylase / trypsin inhibitor encoded by the rice gene OsRAL5, the amino acid sequence of which is shown in SEQ ID NO.2, with a total of 178 amino acids.

[0008] The present invention also provides a gene editing vector of the rice gene OsRAL5, wherein the gene editing vector comprises the nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4 and a pYLCRISPR / Cas9 gene editing vector.

[0009] The present invention also provides an expression vector of the rice gene OsRAL5, which comprises the nucleotide sequence shown in SEQ ID NO.1 and the pUbi-RFP-HA expression vector.

[0010] The invention also provides application of the rice gene OsRAL5 in improving rice grain size and thousand-grain weight.

[0011] The present invention also provides application of the rice gene OsRAL5 in improving the appearance quality and taste quality of rice.

[0012] The present invention also provides the application of the rice gene OsRAL5 in synergistically improving rice variety yield and rice quality.

[0013] Compared with the prior art, the present invention has the following technical effects:

[0014] 1. The present invention discloses for the first time the sequence of the rice allergen protein gene OsRAL5 and the protein sequence encoded by it. qRT-PCR expression analysis shows that the OsRAL5 gene is specifically and highly expressed in the endosperm. The gene can synergistically improve the yield and quality of rice, and can be used as a target gene to be over-expressed in rice, thereby increasing the size and yield of rice grains, improving the appearance quality and taste quality of rice, and providing good gene resources for cultivating high-yield and high-quality rice varieties.

[0015] 2. The present invention discloses for the first time the function of the rice OsRAL5 gene in synergistically positively regulating rice yield and quality. By analyzing the grain shape, endosperm phenotype and rice quality index of the OsRAL5 gene functional deletion edited mutant and overexpression plants, it was found that compared with the wild type, the OsRAL5 gene edited mutant showed phenotypes such as smaller grains, reduced thousand-grain weight, white core endosperm, abnormal starch granule development, and reduced amylose content, while the OsRAL5 gene overexpression plants showed excellent phenotypes such as enlarged grains, good appearance quality, increased amylose content, and increased taste quality compared with the wild type. The rice OsRAL5 gene is involved in the formation of grain size and endosperm starch granules at the same time, and therefore is expected to be used in the synergistic improvement of rice yield and quality.

[0016] 3. The present invention discloses for the first time that the functional loss and overexpression of rice OsRAL5 gene can affect the expression of storage protein and starch synthesis-related genes, and the changes in gene expression levels are consistent with the changes in seed storage protein content.

[0017] 4. The present invention discloses for the first time that the rice OsRAL5 gene has a certain complementary effect on gene expression with homologous genes OsRAG1, OsRAG2, OsRAL2, and OsRAL6. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a plant and grain phenotype analysis diagram of the rice OsRAL5 gene-edited mutant in an embodiment of the present invention. Figure 1 Middle, A, C: Plant phenotypes (A) and grain phenotypes (C) of the wild type and the edited mutants during the filling period; B, DG: Statistical analysis of the number of ears (B), 1000-grain weight (D), grain length (E), grain width (F) and grain thickness (G) of the wild type and the edited mutants.

[0019] Figure 2 This is an analysis diagram of the endosperm phenotype and quality index of the OsRAL5 gene-edited mutant in the embodiment of the present invention. Figure 2Middle, A: endosperm appearance and starch granule structure of wild type and edited mutant, Bar=10um; BE: statistical analysis of total starch content (B), amylose content (C), gel consistency (D) and RVA viscosity (E) of polished rice of wild type and edited mutant.

[0020] Figure 3 It is a sequence analysis diagram of the editing site of the OsRAL5 gene editing mutant in the embodiment of the present invention. Figure 3 In the figure, A: the location of the two editing sites on the OsRAL5 gene; B: sequence analysis of the editing sites TS1 and TS2; C: the CRISPR / Cas9 vector framework used for gene editing.

[0021] Figure 4 This is an expression profile analysis diagram of the OsRAL5 gene in different developmental stages and tissue parts of Nanjing Jinggu in the embodiment of the present invention. Total RNA was extracted from callus, roots and buds 7 days after germination, leaves, stems, panicles, anthers and ovaries before flowering, seeds at different developmental stages, and endosperms and embryos. ACTIN1 was used as an internal reference.

[0022] Figure 5 It is an analysis diagram of the grain shape and endosperm phenotype and yield and quality indexes of the OsRAL5 gene overexpressing plant in the embodiment of the present invention. Figure 5 A: grain shape phenotype of wild-type and overexpression plants; B: endosperm appearance of wild-type and overexpression plants; CF: statistical analysis of thousand-grain weight (C), amylose content (D), rice appearance (E) and taste value (F) of wild-type and overexpression plants.

[0023] Figure 6 This is an expression analysis diagram of genes related to seed size, protein and starch synthesis in OsRAL5 gene-edited and overexpressed plants in the examples of the present invention.

[0024] Figure 7 It is an evolutionary analysis diagram of the OsRAL5 protein in the examples of the present invention. Figure 7 In the figure, the black boxes indicate five genes of the α-amylase / trypsin inhibitor family.

[0025] Figure 8 This is a graph of expression analysis of α-amylase / trypsin inhibitor family genes in seeds 14 days after flowering of OsRAL5 gene-edited and overexpressed plants in an example of the present invention. DETAILED DESCRIPTION

[0026] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments.

[0027] Example 1: Mutation of the rice gene OsRAL5 leads to smaller grains, lower thousand-grain weight, white endosperm, and abnormal starch granule development:

[0028] From the rice gene editing mutant library created with the excellent japonica rice variety Nanjing Jinggu as the background, the materials dOsRAL5-TS1 and dOsRAL5-TS2 with two sites edited on the allergen protein gene OsRAL5 were screened. Through the analysis of agronomic traits, it was found that the plant height and panicle number of OsRAL5-edited plants were not significantly different from those of the control (Nanjing Jinggu, NJJG), while the thousand-grain weight, grain length, grain width and grain thickness were significantly reduced compared with the control ( Figure 1 ).

[0029] Further analysis of rice quality indicators revealed that the grains of the edited plants dOsRAL5-TS1 and dOsRAL5-TS2 showed core white endosperm, and scanning electron microscopy showed that the starch granules in the core white part were abnormally developed and presented irregular spherical shapes. The physical and chemical index tests of rice revealed that the total starch content of the edited plants increased, the amylose content and gel consistency decreased significantly, and the RVA spectrum viscosity also decreased significantly ( Figure 2 ).

[0030] Example 2: Editing site sequence analysis of OsRAL5 gene-edited plants:

[0031] Both editing sites are located on the exon of the OsRAL5 gene, and the targeting sequences are 20bp conservative specific sequences. Target 1 (sgRNA1) is located at 153-172bp of the OsRAL5 gene, and the nucleotide sequence is SEQ ID NO.3; Target 2 (sgRNA2) is located at 192-211bp of the OsRAL5 gene, and the nucleotide sequence is SEQ ID NO.4( Figure 3 A). Editing site sequence analysis revealed that the dOsRAL5-TS1 plant had a 7bp deletion at the TS1 site, and the dOsRAL5-TS2 plant had an 11bp deletion at the TS2 site ( Figure 3 B), Both editing events resulted in frameshift mutations.

[0032] The construction of the targeting vector is mainly based on the operating steps of (Ma et al, Molecular Plant, 2015, 8 (8), 1274-1284). According to the requirements of the U3 vector, the U3-sgRNA-F / R linker is synthesized; the sgRNA is connected to the OsU3 plasmid vector by preparing oligo dimers, and the DNA fragment of the U3-sgRNA is obtained by two rounds of PCR amplification; it is then connected to the expression vector pYLCRISPR / Cas9, and finally the editing vectors containing different targets are constructed, such as Figure 3C. Fluorescence quantitative RT-PCR detection showed that the expression level of OsRAL5 gene was significantly inhibited in the two edited plants ( Figure 8 ).

[0033] Example 3: OsRAL5 gene is specifically expressed in rice endosperm:

[0034] In order to comprehensively analyze the biological function of the allergen protein gene OsRAL5, the temporal and spatial expression pattern of the OsRAL5 gene in wild-type Jingu rice was first studied using qRT-PCR technology. Figure 4 As shown in the results, the expression level of OsRAL5 was extremely low and almost undetectable in callus tissue, vegetative tissues such as roots and buds 7 days after germination, leaves, stems, panicles, stamens and pistils at the booting stage. The expression level was also low in seeds at the early stage of filling. However, with the development and filling process of seeds, the expression level of OsRAL5 increased rapidly and reached the peak in the endosperm 25 days after fertilization. This expression pattern was highly consistent with the filling process of rice endosperm. In addition, through more detailed analysis, it was found that OsRAL5 was mainly expressed in the endosperm, while the expression level in the embryo was extremely low, which was an endosperm-specific expression gene. These results suggest that OsRAL5 plays an important role in seed development.

[0035] Example 4: Overexpression of OsRAL5 leads to larger grains, improved appearance quality, and increased taste quality:

[0036] In order to further verify the biological function of OsRAL5 in rice grain and starch granule development, an overexpression vector of OsRAL5 gene was constructed, and the full-length cDNA of OsRAL5 (including the promoter region of about 2kb above the start codon ATG, the ORF region, and the terminator region of about 1kb downstream of the stop codon) was amplified with specific primers and cloned into the pUbi-RFP-HA vector under the control of the maize ubiquitin promoter (ZmUbi1). Fluorescence quantitative RT-PCR detection showed that the expression level of OsRAL5 gene was significantly increased ( Figure 8 ), indicating that OsRAL5 overexpressing transgenic plants were successfully constructed.

[0037] The grain phenotype and physicochemical indexes of the taste quality of the overexpressing transgenic plants were then analyzed. The results showed that compared with the wild type, the grain shape (grain length and width) of the overexpressing transgenic plants was significantly larger, the thousand-grain weight was significantly increased, the endosperm was transparent, and there was no chalky phenotype; the amylose content of rice was significantly increased, and the appearance quality and taste of the rice were also significantly improved ( Figure 5 ). In summary, the rice gene OsRAL5 positively regulates rice grain shape, amylose content and taste quality.

[0038] Example 5: Analysis of total protein and component contents in OsRAL5 gene-edited and overexpressed plants:

[0039] In order to analyze the effect of OsRAL5 gene on the accumulation of seed storage proteins and their components, the total protein and its components in seeds of OsRAL5 gene-edited and overexpressed plants were analyzed and measured (Table 1). The results showed that compared with the wild type, the total protein content was significantly decreased in OsRAL5-edited plants, while it was significantly increased in overexpressed plants. In the analysis of the content of four protein components, compared with the wild type, the content of glutenin and albumin was significantly decreased in OsRAL5-edited plants, while it was significantly increased in overexpressed plants; while the content of alcohol-soluble protein was significantly increased in OsRAL5-edited plants, significantly decreased in overexpressed plants, and the content of globulin did not change significantly. The allergenic protein OsRAL5 belongs to the albumin gene family. In summary, the editing of OsRAL5 gene led to a decrease in the content of albumin and glutenin, while an increase in the content of globulin and alcohol-soluble protein.

[0040] Table 1 Protein content in rice endosperm

[0041]

[0042] Example 6: Analysis of expression levels of genes related to grain shape and storage substances in OsRAL5 gene-edited and overexpressed plants:

[0043] In order to verify the role of OsRAL5 in regulating seed size and seed storage materials, seeds of OsRAL5 gene-edited and overexpressed plants were selected on the 14th day after flowering, seed RNA was extracted, and the expression levels of related genes were detected using reverse transcription products. Figure 6 As shown, the expression levels of grain shape-related genes (GS3, GS5, GL6, GL7) did not change significantly between edited plants and overexpressed plants; the expression levels of seed glutenin genes (GluA, GluB) decreased in edited plants and increased in overexpressed plants, and the expression levels of seed alcohol-soluble protein genes (Prol14, RP10) increased in edited plants and decreased in overexpressed plants; the expression level of granule-bound starch synthase gene GBSSI decreased in edited plants and increased in overexpressed plants, and other starch synthesis-related genes (OsAGPL2, SSI, SSIIa) all showed an increase in expression in edited plants and a decrease in expression in overexpressed plants. This indicates that the effect of OsRAL5 on seed storage material content is consistent with the expression of related genes. OsRAL5 overexpression can increase the protein content of rice seeds, reduce the starch content, and improve the nutritional quality of seeds. The regulatory mechanism of OsRAL5 affecting grain shape and yield is not included in the existing molecular biological pathways for regulating grain shape and yield.

[0044] Example 7: Evolution analysis of rice OsRAL5 protein:

[0045] In order to clarify the homologous genes and evolutionary relationships of OsRAL5 in rice and other species, the amino acid sequence of OsRAL5 was subjected to BLAST analysis in NCBI, and 28 annotated genes were found. Among them, the rice RAs gene family was clustered into one category, including 5 homologous genes: OsRAG1, OsRAG2, OsRAL2, OsRAL5 and OsRAL6. Other closely related sequences were mainly homologous genes from monocots such as barley, wheat, and sorghum ( Figure 7 ).

[0046] Example 8: Gene expression analysis of rice α-amylase / trypsin inhibitor family (OsRAL5 homologous gene):

[0047] In order to clarify the relationship between rice OsRAL5 and its homologous genes, the expression levels of five genes of the α-amylase / trypsin inhibitor family were detected in OsRAL5-edited plants and overexpressed plants. The expression levels of four OsRAL5 homologous genes, OsRAL6, OsRAG1, OsRAL2, and OsRAG2, were significantly upregulated in OsRAL5-edited plants, but decreased in overexpressed plants ( Figure 8 ), indicating that there is a certain complementary effect between homologous genes.

[0048] Primers and sequences used in the above examples:

[0049]

[0050]

[0051] The above are only implementation modes of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent structure made using the contents of the present invention specification, directly or indirectly used in other related technical fields, is also within the patent protection scope of the present invention.

Claims

1. Application of rice gene OsRAL5 in increasing rice grain size and 1000-grain weight, the nucleotide sequence of the rice gene OsRAL5 is shown in SEQ ID NO.

1.

2. Application of rice gene OsRAL5 in improving rice appearance quality and taste quality. The nucleotide sequence of the rice gene OsRAL5 is shown in SEQ ID NO.

1.

3. Application of the rice gene OsRAL5 in synergistically improving rice variety yield and rice quality, the nucleotide sequence of the rice gene OsRAL5 is shown in SEQ ID NO.1.

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