Regulation of rice gelatinization temperature gene osglip11 and its encoded protein and application

Abstract

The application belongs to the field of plant breeding and biotechnology, and particularly relates to how to reduce the gelatinization temperature of rice by gene OsGLIP11 and application of the gene in creating new germplasm of rice with low gelatinization temperature. The application discloses a nucleic acid sequence of a coding region of gene OsGLIP11, a protein sequence coded by the gene, a target site nucleic acid sequence of CRISPR-Cas9 knockout, a nucleic acid sequence of a knockout mutant and a protein sequence of the knockout mutant, as shown in a standard nucleotide or amino acid sequence table of ST.26; after the gene OsGLIP11 is knocked out by using CRISPR-Cas9 technology, it is found that the gelatinization temperature of rice of the transgenic plant with the gene OsGLIP11 knocked out is significantly lower than that of a wild type variety; after the transgenic plant with the gene OsGLIP11 knocked out is subjected to genetic function complementation by using transgenic function complementation technology, the gelatinization temperature of rice of the function complementation transgenic plant has no significant difference with that of the wild type variety. The application can create new germplasm of rice with low gelatinization temperature by using the gene OsGLIP11.

Description

Technical Field

[0001] This invention belongs to the field of plant breeding and biotechnology, specifically involving genes that regulate rice gelatinization temperature. OsGLIP11 Its encoded proteins and applications. Background Technology

[0002] Rice ( Rice Rice (L.) is one of the most important food crops, with more than half of the world's population relying on it as a staple food. In recent years, the demand for high-quality rice has become increasingly urgent. Rice quality mainly includes cooking and eating quality, appearance quality, milling and processing quality, and nutritional quality. Among these, cooking and eating quality is the most important indicator of rice's commercial value, and gelatinization temperature is one of the main indicators for evaluating rice's cooking and eating quality. Therefore, elucidating the genetic mechanism of rice gelatinization temperature is one of the keys to achieving high-quality rice breeding, and will provide a theoretical basis for high-quality, high-yield, and stable-yield bio-breeding of rice and other crops. Rice gelatinization temperature is jointly regulated by one major gene and several minor genes, and its genetic mechanism is very complex and still needs further refinement. Studies have found that both starch (the most abundant component) and lipids (the least abundant component) in polished rice affect gelatinization temperature. Currently, several genes related to starch's influence on gelatinization temperature have been cloned, but the genetic mechanism of lipids' influence on gelatinization temperature has been rarely reported. To date, no GDSL lipase encoding gene has been identified. OsGLIP11 Reports on regulating the gelatinization temperature of rice. Summary of the Invention

[0003] In view of this, the present invention addresses the problems existing in current research on rice gelatinization temperature by disclosing a gene that regulates rice gelatinization temperature. OsGLIP11 This gene encodes a GDSL lipase, which has been disclosed. OsGLIP11 The coding region sequence of the gene and the sequence of the protein it encodes are shown in SEQ ID NO. 1 and SEQ ID NO. 2 of the ST.26 standard sequence nucleotide or amino acid sequence listing. This invention discloses that the gene can be knocked out... OsGLIP11 By altering the OsGLIP11 protein through gene modification, new rice germplasm with lower gelatinization temperatures can be created, which has significant application value for improving rice quality. Attached Figure Description

[0004] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings: Figure 1 This invention utilizes CRISPR-Cas9 gene editing technology to knock out... OsGLIP11 Genetic technology methods. Including OsGLIP11Information on a target site selected in the gene coding region and the nucleotide mutation types of the two homozygous knockout mutants obtained, as well as the resulting amino acid changes. NIP is wild-type (Nipponbare), and Osglip11-1 and Osglip11-2 are two homozygous knockout mutants; Figure 2 shows the knockout mutants obtained by the method of this invention. OsGLIP11 Gelatinization temperatures of gene mutants, wild-type, and transgenic homozygous complementary lines. WT represents wild-type (Nipponbare), Cr-osglip11-1 and Cr-osglip11-2 are two homozygous knockout mutants, CP-1 is a transgenic homozygous complementary line, To is the initial gelatinization temperature, Tp is the peak gelatinization temperature, Tc is the final gelatinization temperature, and ΔH is the gelatinization enthalpy. ** indicates significant (P < 0.01); Figure 3 shows the knockout results obtained using the method of this invention. OsGLIP11 Photographs of the main agronomic traits of the gene mutants and wild types. NIP is the wild type (Nipponbare), and Cr-osglip11-1 and Cr-osglip11-2 are two homozygous knockout mutants. Detailed Implementation

[0005] This invention discloses a method utilizing OsGLIP11 The application method of regulating rice gelatinization temperature using genes and their encoded proteins includes the following implementation steps: 1) Using CRISPR-Cas9 gene editing technology to... OsGLIP11 Gene knockout is performed. When constructing the CRISPR-Cas9 knockout vector, select... OsGLIP11 The forward nucleotide sequence of a target site in the gene coding region is 5′→3′: as shown in SEQ ID No. 3 of the standard sequence nucleotide or amino acid sequence listing of ST.26; 2) Obtaining knockout OsGLIP11 Homozygous knockout mutant lines of the gene. After constructing the knockout vector, a conventional japonica rice variety, Nipponbare, was selected as the transformation background for genetic transformation, resulting in two homozygous knockout mutant transgenic lines. One of the knockout mutants... OsGLIP11 The forward nucleotide sequence of the gene coding region is 5′→3′: as shown in SEQ ID No. 4 of the ST.26 standard sequence nucleotide or amino acid sequence listing, while the forward amino acid sequence is 5′→3′: as shown in SEQ ID No. 6 of the ST.26 standard sequence nucleotide or amino acid sequence listing; another type of knockout mutant OsGLIP11The forward nucleotide sequence of the gene coding region is 5′→3′: as shown in SEQ ID No. 5 of the ST.26 standard sequence nucleotide or amino acid sequence listing. The forward amino acid sequence is 5′→3′: as shown in SEQ ID No. 7 of the ST.26 standard sequence nucleotide or amino acid sequence listing; 3) Obtain a transgenic functional complementation homozygous line of the OsGLIP11 gene knockout mutant. Functional complementation of one of the knockout mutants in step 2) was verified using transgenic functional complementation experimental techniques, and a homozygous OsGLIP11 gene transgenic functional complementation homozygous line was obtained; 4) Determine the wild-type and knockout... OsGLIP11 Gelatinization temperature and major agronomic traits in rice from transgenic mutants and homozygous lines of functionally complementary transgenic genes. Differential scanning calorimetry (DSC) was used to determine gelatinization temperature, revealing that knockout genes... OsGLIP11 The rice from the mutant gene had a significantly lower gelatinization temperature than the wild type, while plant height and grain shape showed no significant difference from the wild type. OsGLIP11 The rice gelatinization temperature of homozygous transgenic lines with gene functional complementation differed significantly from their transformation background. This invention provides a novel gene that regulates rice gelatinization temperature. OsGLIP11 The OsGLIP11 gene, along with its encoded protein and applications, positively regulates rice gelatinization temperature; knocking out this gene can significantly reduce rice gelatinization temperature.

Claims

1. Knock out the gene for rice gelatinization temperature OsGLIP11 Its application in lowering the gelatinization temperature of rice is characterized by, The OsGLIP11 The accession number of the gene in the rice genome is LOC_Os01g42730 The nucleic acid sequence of its coding region and the protein sequence it encodes are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

2. A method utilizing the method described in claim 1 OsGLIP11 The application of genes in lowering rice gelatinization temperature is characterized by, By using CRISPR-Cas9 gene editing technology to knock out OsGLIP11 Genes are used to lower the gelatinization temperature of rice.

3. The application according to claim 2, characterized in that, When constructing the CRISPR-Cas9 knockout vector, select OsGLIP11 A target site on a gene coding region, wherein the forward nucleotide sequence of the target site is 5′→3′: as shown in SEQ ID No.

3.

4. The application according to claim 2, characterized in that, Two gene editing techniques were obtained using CRISPR-Cas9 gene editing technology. OsGLIP11 Gene knockout mutants, one type of knockout mutant OsGLIP11 The forward nucleotide sequence of the gene coding region is 5′→3′: as shown in SEQ ID No. 4, another type of knockout mutant. OsGLIP11 The forward nucleotide sequence of the gene coding region is 5′→3′, as shown in SEQ ID No.

5.

5. The application according to claim 2, characterized in that, Two gene editing techniques were obtained using CRISPR-Cas9 gene editing technology. OsGLIP11 Gene knockout mutants, one of which has a forward amino acid sequence of 5′→3′ as shown in SEQ ID No. 6, and the other has a forward amino acid sequence of 5′→3′ as shown in SEQ ID No. 7.

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