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CN-121975761-A - Rice SBEIIb mutant protein and application thereof in improving rice quality

CN121975761ACN 121975761 ACN121975761 ACN 121975761ACN-121975761-A

Abstract

The invention belongs to the field of rice genetic breeding, and relates to a rice SBEIIb mutant protein and application thereof in improving rice quality. By constructing a CBE vector targeting OsSBEIIb gene 12 exon specific sequence, the OsSBEIIb protein 446 th glycine G undergoes three different forms of substitution mutation including G446R, G446K and G446T by using glutinous rice Guanglingxiang as a transformation receptor material. The starch components, thermodynamic properties and viscous properties of the three allelic mutant rice with single amino acid substitution are all obviously changed, and the allelic mutant rice has different application potentials. The three homozygous OsSBEIIb gene allelic mutants without exogenous genes provided by the invention are valuable germplasm resources, and provide an effective strategy for rapidly improving rice starch components and cultivating high-quality functional new rice strains.

Inventors

  • Ke Minxue
  • WEI CUNXU
  • Shi Laiquan
  • CHEN MENGYA
  • FENG YUANHAO
  • SHENG WENJING
  • ZHANG LONG

Assignees

  • 扬州大学

Dates

Publication Date
20260505
Application Date
20251226

Claims (10)

  1. 1. The rice SBEIIb mutant protein is characterized in that the 446 th glycine G of the SBEIIb protein coded by OsSBEIIb genes is substituted and mutated into one of T, R and K, the nucleotide sequence of OsSBEIIb genes is shown as SEQ ID NO.1, and the coded protein sequence is shown as SEQ ID NO. 2.
  2. 2. The rice SBEIIb mutant protein according to claim 1, The amino acid sequence of the rice SBEIIb mutant protein with the 446 th glycine G of the SBEIIb protein being mutated into R is shown as SEQ ID NO. 3; the amino acid sequence of the rice SBEIIb mutant protein with the 446 th glycine G of the SBEIIb protein being mutated into K is shown as SEQ ID NO. 4; The amino acid sequence of the rice SBEIIb mutant protein with the 446 th glycine G of the SBEIIb protein being mutated into T is shown as SEQ ID NO. 5.
  3. 3. Use of the rice SBEIIb mutant protein according to claim 1 for improving rice quality.
  4. 4. The use according to claim 3, wherein the improvement of rice quality comprises improvement of starch component, thermodynamic properties and viscosity properties of rice.
  5. 5. A method for improving the quality of rice, comprising the steps of: designing OsSBEIIb target sites for gene site-specific editing; Constructing a single base editing vector containing a target fragment; Obtaining a T 0 transgenic rice plant, namely transforming agrobacterium tumefaciens EHA105 by a single base editing vector, and obtaining a T 0 transformed plant by an agrobacterium-mediated rice genetic transformation method; Screening T 1 mutant plants without exogenous genes, and obtaining target gene homozygous mutant plants to obtain the rice material for improving the rice quality.
  6. 6. The method for improving rice quality according to claim 1, wherein the target site comprises a target sequence of sgRNA, wherein the target sequence of sgRNA is a base sequence shown as SEQ ID NO.6, and corresponds to the base sequence of nucleotide numbers 104-123 of exon 12 of OsSBEIIb gene.
  7. 7. The method for improving rice quality according to claim 1, wherein screening the exogenous gene-free T 1 mutant plants comprises PCR amplification and identification of sgRNA, wherein the identification primers are shown as SEQ ID NO.9 and SEQ ID NO. 10.
  8. 8. The method for improving rice quality according to claim 1, wherein screening the exogenous gene-free T 1 mutant plants comprises PCR amplification identification of HPT elements and Cas9 elements, wherein the identification primers are shown in SEQ ID NO. 13-16.
  9. 9. The method for improving rice quality according to claim 1, wherein screening the T 1 mutant plants without the foreign gene comprises PCR amplification identification of the target site fragment, wherein the identification primers are shown as SEQ ID NO.11 and SEQ ID NO. 12.
  10. 10. The method for improving rice quality according to claim 1, wherein the nucleotide sequences of the linker sequences sgRNA-SBEIIb-F and sgRNA-SBEIIb-R of the desired fragment are shown in SEQ ID NO.7 and SEQ ID NO. 8.

Description

Rice SBEIIb mutant protein and application thereof in improving rice quality Technical Field The invention belongs to the field of rice genetic breeding, and relates to a rice SBEIIb mutant protein and application thereof in improving rice quality. Background Rice is one of the most important grain crops in China, and starch is the most important nutritional ingredient and consists of amylose and amylopectin. The amylose content and the side chain structure of amylopectin directly determine the physicochemical properties of starch and rice quality. For a long time, starch component, gelatinization property and viscosity property have been key indexes for evaluating rice quality. Starch branching enzyme IIb (Starch branching enzyme IIb, SBEIIb) is used as a key enzyme for starch synthesis and is responsible for catalyzing branched short side chains of amylopectin to be formed. It was found that SBEIIb deletion mutations or down-regulation of their expression significantly alter starch characteristics and increase rice resistant starch content, but at the same time also severely deteriorate rice quality, especially appearance quality and cooking taste quality, which limits their use in production practice. At present, researches report that certain SBEIIb amino acid substitutions can effectively regulate the starch component of rice and improve the rice quality, which provides possibility for the gene to become an important genetic resource for rice quality improvement breeding. However, the information about such amino acid substitution mutations is very poor, severely limiting the molecular design breeding process of rice quality. Therefore, the novel SBEIIb mutant gene capable of regulating and controlling the starch component, gelatinization property and viscosity property of rice and the amino acid substitution mutant protein encoded by the novel SBEIIb mutant gene are excavated and created, so that germplasm resources are provided for rice quality improvement and breeding, and the novel SBEIIb mutant gene has important scientific significance and application value for molecular design and breeding for cultivating high-quality rice varieties. Disclosure of Invention In order to solve the technical problems, the invention provides rice SBEIIb mutant protein and application thereof in improving rice quality, wherein the rice SBEIIb mutant protein can obviously improve starch components and functional characteristics of rice. The technical scheme provided by the invention is as follows: The rice SBEIIb mutant protein is one of T, R and K which is obtained by substitution mutation of glycine G at 446 th position of SBEIIb protein coded by SBEIIb gene, the nucleotide sequence of the SBEIIb gene is shown as SEQ ID NO.1, and the coded protein sequence is shown as SEQ ID NO. 2. Furthermore, the amino acid sequence of the rice SBEIIb mutant protein with the 446 th glycine G substitution mutation of the SBEIIb protein being R is shown as SEQ ID NO.3, the amino acid sequence of the rice SBEIIb mutant protein with the 446 th glycine G substitution mutation of the SBEIIb protein being K is shown as SEQ ID NO.4, and the amino acid sequence of the rice SBEIIb mutant protein with the 446 th glycine G substitution mutation of the SBEIIb protein being T is shown as SEQ ID NO. 5. The invention also provides application of the rice SBEIIb mutant protein in improving rice quality. Further, the improving of the rice quality of the rice includes improving a starch component, a thermodynamic property and a viscous property of the rice. The invention also provides a method for improving the quality of rice, which comprises the following steps: designing OsSBEIIb target sites for gene site-specific editing; Constructing a single base editing vector containing a target fragment; Obtaining a T 0 transgenic rice plant, namely transforming agrobacterium tumefaciens EHA105 by a single base editing vector, and obtaining a T 0 transformed plant by an agrobacterium-mediated rice genetic transformation method; Screening T 1 mutant plants without exogenous genes, and obtaining target gene homozygous mutant plants to obtain the rice material with improved rice quality. Furthermore, the target site comprises a target sequence of sgRNA, wherein the target sequence of sgRNA is a base sequence shown as SEQ ID NO.6, and corresponds to the base sequence of the OsSBEIIb th exon of the gene from the 104 th to the 123 th nucleotide. Further, the nucleotide sequences of the linker sequences sgRNA-SBEIIb-F and sgRNA-SBEIIb-R of the target fragment are shown as SEQ ID NO.7 and SEQ ID NO. 8. Further, screening T 1 mutant plants without exogenous genes comprises PCR amplification identification of sgRNA, and the identification primers are shown as SEQ ID NO.9 and SEQ ID NO. 10. Further, screening the T 1 mutant plants without the exogenous genes comprises PCR amplification identification of the HPT element and the Cas9 element, wherein the identification