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CN-120026056-B - Application of rice endosperm storage substance genes

CN120026056BCN 120026056 BCN120026056 BCN 120026056BCN-120026056-B

Abstract

The present disclosure provides application of rice endosperm storage substance genes, and belongs to the field of biotechnology. The application comprises that the RSM gene is used for regulating and controlling the content of rice endosperm storage substances. The embodiment of the invention provides application of a rice endosperm storage substance gene, and RSM gene is used for regulating and controlling the content of rice endosperm storage substance, and has obvious effect.

Inventors

  • SHI SHAOJIE
  • Li sanhe
  • LIU KAI
  • YANG GUOCAI
  • CHEN ZHIJUN
  • LIU KAI
  • XU HUASHAN
  • LI PEIDE
  • YOU AIQING
  • ZHOU LEI
  • WANG HUIYING
  • ZHA WENJUN
  • WU YAN
  • CHEN JUNXIAO
  • LI CHANGYAN
  • WU BIAN

Assignees

  • 湖北省农业科学院粮食作物研究所

Dates

Publication Date
20260508
Application Date
20250220
Priority Date
20240919

Claims (7)

  1. 1. The application of the rice endosperm storage substance gene is characterized in that the application comprises knocking out the amino acid sequence of the RSM gene, wherein the sequence of the RSM gene is shown as SEQ ID NO. 6 in a sequence table, and the amino acid sequence is used for improving the total protein content and the gluten content and reducing the globulin content, the prolamin content and the albumin content.
  2. 2. The application according to claim 1, characterized in that it comprises: Selecting RSM-T from the sequence of the RSM gene as a target point of gene knockout, wherein the sequence of the RSM-T is shown as SEQ ID NO. 1 in a sequence table; Constructing an RSM gene knockout vector according to the RSM-T by adopting a first forward primer, a first reverse primer, a second forward primer and a second reverse primer, wherein the sequence of the first forward primer is shown as SEQ ID NO. 2 in a sequence table, the sequence of the first reverse primer is shown as SEQ ID NO. 3 in the sequence table, the sequence of the second forward primer is shown as SEQ ID NO. 4 in the sequence table, and the sequence of the second reverse primer is shown as SEQ ID NO. 5 in the sequence table; and (3) transforming the RSM gene knockout vector into a receptor material to obtain the RSM gene homozygous knockout mutant.
  3. 3. The use according to claim 2, characterized in that it comprises using pYLsgRNA-OsU a plasmid as template, amplifying by a first PCR with the first forward primer and the first reverse primer to obtain a first amplification product comprising the U6b promoter of the RSM-T, amplifying by a second PCR with the second forward primer and the second reverse primer to obtain a second amplification product comprising the guided sgRNA fragment of the RSM-T; Purifying and recovering the first amplification product and the second amplification product to obtain a purified first amplification product and a purified second amplification product; connecting the purified first amplification product and the purified second amplification product through overlapping PCR to obtain a connection product; And connecting the connecting product to PYLCRISPR/Cas9Pubi-H vector to obtain the RSM gene knockout vector.
  4. 4. The method according to claim 3, wherein the reaction system for the first PCR amplification comprises 10 XPCR Buffer 5. Mu.L, 2 mM dNTP 5. Mu.L, the first forward primer 1.5. Mu.L at a concentration of 10. Mu.M, the first reverse primer 1.5. Mu.L at a concentration of 10. Mu.M, 1. Mu.L of template DNA, 1. Mu.L of KOD-Plus-Neo high-fidelity polymerase at a concentration of 5U/. Mu.L, and ddH 2 O35. Mu.L per 50. Mu.L of the first PCR amplification reaction system.
  5. 5. The use according to claim 3, wherein the reaction system for the second PCR amplification comprises 10 XPCR Buffer 5. Mu.L, 2mM dNTP 5. Mu.L, the second forward primer 1.5. Mu.L at a concentration of 10. Mu.M, the second reverse primer 1.5. Mu.L at a concentration of 10. Mu.M, template DNA 1. Mu.L, KOD-Plus-Neo high-fidelity polymerase 1. Mu.L at a concentration of 5U/. Mu.L, ddH 2 O35. Mu.L per 50. Mu.L.
  6. 6. The use according to claim 3, wherein the reaction system for the overlap PCR comprises 10 XPCR Buffer 5. Mu.L, 2mM dNTP 5. Mu.L, the first forward primer 1.5. Mu.L at a concentration of 10. Mu.M, the second reverse primer 1.5. Mu.L at a concentration of 10. Mu.M, the promoter U6a 0.5. Mu.L, the guide fragment sgRNA 0.5. Mu.L, KOD-Plus-Neo Hi-Fidelity polymerase 1. Mu.L at a concentration of 1U/. Mu.L, ddH 2 O35. Mu.L per 50. Mu.L.
  7. 7. The use according to claim 3, wherein each 15. Mu.L of the ligated reaction system comprises 10X CutSmart Buffer 1.5.5. Mu.L, 1.5. Mu.L of 10mM ATP cocktail, 0.5. Mu.L of 200 ng/. Mu. L PYLCRISPR/Cas9Pubi-H vector, 1. Mu.L of 50 ng/. Mu.L sgRNA expression cassette fragment, 0.5. Mu.L of 20U/. Mu.L BsaI-HF endonuclease, 0.2. Mu.L of 400U/. Mu. L T4 DNA ligase, and 9.8. Mu.L of ddH 2 O.

Description

Application of rice endosperm storage substance genes Technical Field The present disclosure relates to biotechnology, and in particular, to application of rice endosperm storage substance genes. Background Rice is the staple food for more than half of the world population. Breeders have long focused on how to increase rice yield to meet the increasing demand of the population for food. With the progress of breeding technology in recent years, especially the utilization of dwarf breeding and heterosis, the yield of grains is greatly improved. However, as the living standard increases, so does the consumption concept of people, and the state of being fully eaten is changed from eating to eating nutrition and health, and the higher demands are put on the quality of rice. Therefore, the cultivation of new rice varieties with high yield and quality has become the main attack direction of breeders. The rice quality traits mainly include appearance quality, processing quality, steamed taste variety and nutritional quality. Starch and storage proteins are the main components of rice endosperm, wherein starch accounts for more than 80% of the dry weight of endosperm, and starch in endosperm comprises amylose and amylopectin, and a great deal of research has shown that the proportion of starch content, amylose content and amylopectin content in endosperm seriously affects the quality of rice. The protein is the storage substance next to starch in rice endosperm, and accounts for 5% -10% of endosperm dry weight, and can be roughly classified into gluten, albumin, globulin and prolamin 4, and the content and proportion of the gluten, albumin, globulin and prolamin can influence the nutrition, processing, appearance and taste quality of rice. At present, the high-quality breeding of rice is slow, and the reason is that the quality forming mechanism of rice is unclear, so that the quality related genes are less in location cloning, and particularly, the genes related to storage substances are less. Therefore, the development of new storage substance related genes and the construction of related mutants are of great significance for analyzing the rice quality formation mechanism and rice quality breeding. BRIEF SUMMARY OF THE PRESENT DISCLOSURE In order to solve the problems of the prior art, the embodiment of the disclosure provides an application of rice endosperm storage substance genes. The technical scheme is as follows: The disclosure provides an application of a rice endosperm storage material gene, which is characterized in that the application comprises the application of an RSM gene to control the content of rice endosperm storage material. Specifically, the application comprises the steps of knocking out, replacing or changing the amino acid sequence of the RSM gene to reduce the linear-chain light powder content of rice and improve the amylopectin content, and keeping the total starch content of the rice unchanged. In particular, the use includes knocking out, replacing or modifying the amino acid sequence of the RSM gene for increasing total protein content and gluten content, and decreasing globulin content, prolamin content and albumin content. Specifically, the application comprises the steps of selecting RSM-T from the sequence of the RSM gene as a target point of gene knockout, wherein the sequence of the RSM-T is shown as SEQ ID NO. 1 in a sequence table; Constructing an RSM gene knockout vector according to the RSM-T by adopting a first forward primer, a first reverse primer, a second forward primer and a second reverse primer, wherein the sequence of the first forward primer is shown as SEQ ID NO. 2 in a sequence table, the sequence of the first reverse primer is shown as SEQ ID NO. 3 in the sequence table, the sequence of the second forward primer is shown as SEQ ID NO. 4 in the sequence table, and the sequence of the second reverse primer is shown as SEQ ID NO. 5 in the sequence table; and (3) transforming the RSM gene knockout vector into a receptor material to obtain the RSM gene homozygous knockout mutant. Further, the application comprises using pYLsgRNA-OsU a plasmid as a template, amplifying by a first PCR using the first forward primer and the first reverse primer to obtain a first amplification product comprising the U6b promoter of the RSM-T, amplifying by a second PCR using the second forward primer and the second reverse primer to obtain a second amplification product comprising the guided sgRNA fragment of the RSM-T; Purifying and recovering the first amplification product and the second amplification product to obtain a purified first amplification product and a purified second amplification product; connecting the purified first amplification product and the purified second amplification product through overlapping PCR to obtain a connection product; And connecting the connecting product to PYLCRISPR/Cas9Pubi-H vector to obtain the RSM gene knockout vector. Further, each 50 mu L of th