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Mutant OsGBSS1 enzyme and preparation method and application thereof

A technology of A114, starch synthase, applied in the field of enzymology, can solve problems such as hindering application, inability to quantify, and lack of systematic research on OsGBSS1 enzyme activity

Inactive Publication Date: 2016-03-16
CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the inhibitory effect of dsRNAi on gene expression can only be qualitative and cannot be accurately quantified, which also hinders its application in improving the quality of rice breeding to a certain extent.
In addition, the above methods are all based on the theory that the expression of Wx is regulated both transcriptionally and post-transcriptionally, and did not systematically study aspects that directly alter the enzymatic activity of OsGBSS1

Method used

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  • Mutant OsGBSS1 enzyme and preparation method and application thereof
  • Mutant OsGBSS1 enzyme and preparation method and application thereof
  • Mutant OsGBSS1 enzyme and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0192] Example 1. Construction of OsGBSS1 site-directed mutation vector

[0193] In order to study the information of the amino acid positions required for the normal function of OsGBSS1, the present invention (1) deduces the amino acids around the binding site of OsGBSS1 (SEQ ID NO: 1) and the substrate ADPG through homology simulation. Using the resolved EcGS (Escherichia coli glycogen synthase) structure (PDB, 3GUH) as a template, the homologously simulated structure of OsGBSS1 ( figure 1 A); (2) Sequence alignment was carried out. After screening, the following sites were selected as sites for point mutations, specifically including: H236, N265, Y268, N353, R408, E410, K413, and C487.

[0194] The inventors performed site-directed mutations on amino acid positions H236, N265, Y268, N353, R408, E410, C487 and K413, and introduced different amino acid substitutions. The principles of amino acid replacement are: (1) conversion of polar amino acids to non-polar amino acids, ...

Embodiment 2

[0196] Embodiment 2. Obtaining of transgenic plants

[0197] The inventors selected two glutinous rice varieties, Fengnuo and Guangling Xiangnuo, as transformation receptor materials for site-directed mutagenesis. Among them, Fengnuo is a conventionally cultivated glutinous rice variety, and Guangling Xiangnuo is a fragrant japonica rice that was bred through multiple generations by crossing the high-stalk fragrant rice variety Ivory No. 2 used by the Agricultural College of Yangzhou University with the Japanese high-quality medium-japonica variety Zhongguo 45. species [25]. The appearance of the seeds of these two kinds of glutinous rice shows the characteristic of glutinous rice that is obviously opaque. Perform I on the longitudinally cut seeds 2 -KI staining showed that Fengnuo and Guanglingxiangnuo were orange after dyeing. The AC of the two materials are 1.52% and 2.22% ( figure 2 A). After detecting the expression of OsGBSS1 in the mature seeds of Fengnuo and Guan...

Embodiment 3

[0206] Embodiment 3. Determination of specific enzyme activity of OsGBSS1 in transgenic plants

[0207] In order to detect whether the different amino acid mutations of OsGBSS1 affect its enzymatic activity and the degree of influence, the inventors first detected the T 2 OsGBSS1 specific enzyme activity in transgenic homozygous strains with single copy insertion. Among the transgenic plants obtained from the two recipient materials of each site-directed mutagenesis vector and control plasmid, the inventors selected 5 T 2 Generation of T-DNA independent lines, each line selected 5-10 immature seed endosperm obtained from plants to carry out OsGBSS1 specific enzyme activity determination.

[0208] The measurement results showed that compared with the transgenic plants transformed with positive plasmids, the specific enzyme activity of OsGBSS1 in the endosperm of the transgenic plants transformed with 10 site-directed mutation vectors was weakened, ranging from 20% to 90% of th...

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Abstract

The invention discloses granule-bound starch synthase. The synthase mutates in one or more amino acid sites corresponding to an amino acid sequence shown in SEQ ID NO:1, wherein the sites are selected from a lower group and are H236, N265, Y268, N353, R408, E410, K413, C487, A114, EA314-315 and T543. The enzyme specific activity of the synthase is reduced. Thus, the relation between specific amino acid residues and the amylose content is disclosed, and therefore the content of amylose in rice can be adjusted, and then the granule-bound starch synthase can be used for improving the edible quality of rice.

Description

[0001] This application was submitted on March 5, 2013, and the application number is 201310069961.3. It is a divisional application of a Chinese patent application with the title of invention creation titled "mutant OsGBSS1 enzyme and its preparation method and use". technical field [0002] The present invention relates to the field of enzymology. Specifically, the present invention relates to a mutant OsGBSS1 (granule-bound starch synthase 1, 2.4.1.242) and a method for improving amylose content in rice using the enzyme and its application. Background technique [0003] Cereal crops are the main food crops in my country. Among them, rice (Oryza sativa) is an important cereal crop, which is divided into indica and japonica. Indica includes indica and indica glutinous, and japonica includes japonica and japonica glutinous. The main part of rice for human consumption is the starch in the endosperm, and the starch has amylose and amylopectin. The difference between glutinou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12Q1/48C12N15/84A01H5/00A01H5/10C12N15/54C12N15/82
CPCC12N9/1051C12N15/8203C12N15/8245C12Q1/48C12Q1/6895C12Y204/01242
Inventor 蔡秀玲刘德瑞
Owner CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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