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KR-102963582-B1 - Loss-of-function mutant of Fructose-2,6-phosphate kinase1 gene and Fructose-2,6-phosphate kinase2 gene from Oriza sativa and use thereof

KR102963582B1KR 102963582 B1KR102963582 B1KR 102963582B1KR-102963582-B1

Abstract

The present invention relates to a double loss-of-function mutant of the F2KP1 gene and the F2KP2 gene derived from rice and its uses. The composition for producing plants for dry milling according to the present invention can produce transgenic rice with a significantly increased degree of endosperm flouriness compared to a single loss-of-function mutant of the Fructose-2,6-phosphate kinase2 gene by inducing a double loss-of-function mutation of the Fructose-2,6-phosphate kinase1 gene and the Fructose-2,6-phosphate kinase2 gene. In addition, rice flour can be easily processed using a cost-effective dry milling method by utilizing the seeds of the transgenic plant, and thus can be utilized in the manufacture of various rice processed foods.

Inventors

  • 전종성
  • 심수현
  • 이상규

Assignees

  • 경희대학교 산학협력단

Dates

Publication Date
20260512
Application Date
20230823

Claims (17)

  1. A composition for producing grains with reduced hardness, comprising an agent that inhibits the expression of the Fructose-2,6-phosphate kinase 1 gene and an agent that inhibits the expression of the Fructose-2,6-phosphate kinase 2 gene, The above composition induces grain hardness reduced by 70-80% compared to the wild type, and The above composition is a composition for producing grains with reduced hardness, which maintains the content of damaged starch in the grains at 4 to 6%.
  2. In paragraph 1, A composition for producing grains with reduced hardness, characterized in that the gene encoding Fructose-2,6-phosphate kinase1 is represented by the nucleotide sequence of SEQ ID NO. 1.
  3. In paragraph 1, A composition for producing grains with reduced hardness, characterized in that the gene encoding Fructose-2,6-phosphate kinase2 is represented by the nucleotide sequence of SEQ ID NO. 2.
  4. In paragraph 1, A composition for producing grains with reduced hardness, characterized in that the above preparation is an antisense oligonucleotide, siRNA, shRNA, miRNA, PNA (protein nucleic acid), ribozyme, CRISPR/Cas9, T-DNA, or DNAzyme.
  5. In paragraph 1, The above composition is a composition for producing grains with reduced hardness, characterized by inducing an opaque endosperm expression trait of the seed.
  6. A method for producing grains with reduced hardness, comprising the step of reducing the activity of the Fructose-2,6-phosphate kinase1 gene and the Fructose-2,6-phosphate kinase2 gene.
  7. In paragraph 6, A method for producing grains with reduced hardness, characterized in that the above step is performed by substituting or deleting some bases of the Fructose-2,6-phosphate kinase1 gene and the Fructose-2,6-phosphate kinase2 gene.
  8. In paragraph 6, A method for producing grains with reduced hardness, characterized in that the above steps are performed by a CRISPR/Cas9 system.
  9. delete
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  14. Grains with reduced hardness produced by the method of paragraph 6.
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  16. Powder produced from the grains of Paragraph 14.
  17. In Paragraph 16, The above powder is a powder produced by dry milling.

Description

Loss-of-function mutant of Fructose-2,6-phosphate kinase1 gene and Fructose-2,6-phosphate kinase2 gene from Oriza sativa and use thereof The present invention relates to double loss-of-function mutants of the rice-derived F2KP1 gene and F2KP2 gene and their uses. Recently, as the rice consumption environment in Korea has changed rapidly due to household restructuring and the pursuit of convenience, the demand for rice for processing has continued to increase. Even excluding rice for alcohol production, approximately 400,000 tons are consumed annually for processing. Of this, 280,000 tons are used in the form of rice flour. Of this 280,000 tons, 224,000 tons are consumed in the form where processing companies mill the rice themselves to produce products, while the volume of rice flour directly distributed is only 56,000 tons. Consequently, the scale of the rice flour milling industry, which serves as an intermediate good for the production of processed foods, is very weak. To revitalize the rice processing industry, a foundation must be established where rice flour is distributed in large supermarkets and department stores like wheat flour, and where various grades of rice flour are supplied according to quality standards for each processed product category, enabling processors and consumers to easily purchase and use it. However, due to the hardness of rice grains (grain hardness), the traditionally used wet milling method acts as the biggest limiting factor for the industrialization of rice flour. This is because wet milling, which involves grinding after soaking in water, is not only more expensive than dry milling but also requires significant investment in establishing facilities for mass production due to its complex process. The most active means of resolving the imbalance in the supply and demand of rice for consumption is the consumption of new rice. However, due to the high cost of wet milling, small-scale processing companies with limited capacity for facility investment and technology development prefer cheap old rice provided by the government or low-quality imported rice over new rice to reduce costs. Therefore, the need to develop rice varieties specifically for dry milling suitable for rice flour has been consistently raised to expand rice consumption through the revitalization of the processing industry. Accordingly, the inventors confirmed that when a double loss-of-function mutation of the F2KP1 gene and the F2KP2 gene is induced in rice, the grain hardness, damaged starch content, amylose content, and total starch content of the seeds are significantly reduced in the double loss-of-function mutant, and thus completed the present invention. Figure 1 is a figure showing the results of comparing the amino acid sequences of OSF2KP1 and OSF2KP2 in one embodiment of the present invention. FIG. 2 is a figure showing the results of the analysis of OsF2KP1 and OsF2KP2 gene expression in one embodiment of the present invention. Figure 3 is a figure showing the results of an expression test of OsF2KP1 and OsF2KP2 according to hypoxic stress treatment in one embodiment of the present invention. FIG. 4 is a schematic diagram of the genotype of a CRISPR/Cas-mediated osf2kp1 osf2kp2 double mutant (f2kp-d) in one embodiment of the present invention. Figure 5 is a figure showing the results of confirming the seed endosperm phenotypes of OsF2KP1 and OsF2KP2 single and double mutants in one embodiment of the present invention. Figure 6 is a figure showing the results of the analysis of starch-related indicators of OsF2KP1 and OsF2KP2 single and double mutations in one embodiment of the present invention. FIG. 7 is a figure showing the results of the analysis of F2KP enzyme activity of OsF2KP1 and OsF2KP2 single and double mutants in one embodiment of the present invention. FIG. 8 is a figure showing the mechanism of action of the F2KP enzyme in the process of rice endosperm starch synthesis in one embodiment of the present invention. Hereinafter, the present invention will be described in detail with reference to the attached drawings and embodiments thereof. However, the following embodiments are presented as examples of the present invention, and if it is determined that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the essence of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the claims set forth below and the equivalent scope interpreted therefrom. Furthermore, the terminology used in this specification is used to appropriately describe preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the conventions of the field to which the present invention belongs. Accordingly, the definitions of these terms should be b