A kind of adp-glucose pyrophosphorylase mutant and its screening method and application

A technology of phosphorylase mutants and glucose pyrolysis, applied in biochemical equipment and methods, applications, botany equipment and methods, etc., can solve problems such as insufficient attention to research on high-starch varieties

Active Publication Date: 2020-07-07
CROP RES INST GUANGDONG ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in order to solve the problem of food shortage, the focus of my country's sweet potato breeding work has been on cultivating high-yield varieties for a long time in the past, and insufficient attention has been paid to the research of high-starch varieties. The starch content of sweet potato varieties is still at a low level, and the starch yield There is still a lot of room for improvement

Method used

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  • A kind of adp-glucose pyrophosphorylase mutant and its screening method and application
  • A kind of adp-glucose pyrophosphorylase mutant and its screening method and application
  • A kind of adp-glucose pyrophosphorylase mutant and its screening method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Embodiment 1: prepare the prokaryotic expression vector of the large and small subunit cDNA recombinant gene of AGPase

Embodiment 1a

[0080] Example 1a: Obtaining of large and small subunit cDNA of corn endosperm and sweet potato AGPase

[0081] (1) Primer design

[0082] According to the cDNA sequences of the large subunit Shrunken-2 and the small subunit gene Brittle-2 of maize endosperm AGPase (GenBank numbers are: M81603, AF334959) and the cDNA sequences of the large and small subunits of sweet potato AGPase (GenBank numbers are: AB271013.1, AB271014 .1, AB271015.1, AB271016.1, AB271011.2, AB271012.1) designed RT-PCR primers; at the same time, in order to facilitate the construction of the vector, a special design was made when designing the primers: 5 The recognition site of restriction endonuclease Nde I is added at the ' end, and the recognition site of restriction endonuclease Kpn I is added at the 3' end; the recognition site of restriction endonuclease Nco I is added at the 5' end of the small subunit, 3' The restriction endonuclease Sac I recognition site was added at the end, and the primer sequ...

Embodiment 1b

[0137] Embodiment 1b: Construction of AGPase large and small subunit cDNA prokaryotic expression vector

[0138] After the AGPase large subunit Shrunken-2 cDNA was double-digested with restriction endonucleases NdeI and Kpn I, it was ligated with the prokaryotic expression vector that had been double-digested with the same enzymes to obtain a recombinant containing the AGPase large subunit Shrunken-2 cDNA Carrier; the sweet potato small subunit SI cDNA was double-digested with restriction endonucleases Nco I and Sac I, and then connected with the recombinant vector of the above-mentioned AGPase large subunit Shrunken-2 at a molar ratio of 1:1 to obtain AGPase-containing The prokaryotic expression vector of the recombinant gene of the large and small subunit cDNA, referred to as SSI;

[0139] After the AGPase large subunit Shrunken-2 cDNA was double-digested with restriction endonucleases NdeI and Kpn I, it was ligated with the prokaryotic expression vector that had been double...

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Abstract

The invention provides an ADP-glucose pyrophosphorylase mutant and a screening method and application thereof. The screening method includes the steps of firstly, synthesizing first chain cDNA; secondly, performing PCR amplification; thirdly, building AGPase large and small subunit cDNA prokaryotic expression vectors; fourthly, performing active screening. The method has the advantages that distant hybridization is performed on AGPase large subunit genes and small subunit genes from different species, and the recombinant gene expression vectors are obtained in a highly-controllable manner; escherichia coli glgC mutant strains are converted and inoculated to a Conberg enrichment solid culture medium, and iodine-potassium iodide dyeing is used to screen the high-activity ADP-glucose pyrophosphorylase mutant. The screening method is simple and practical and stable and reliable. The enzyme activity and glycogen content of the ADP-glucose pyrophosphorylase mutant are evidently higher than those of wild corn AGPase, evident change of the substrate affinity of the ADP-glucose pyrophosphorylase mutant is avoided, and the sensitivity of the ADP-glucose pyrophosphorylase mutant to activating agent is increased.

Description

technical field [0001] The invention relates to an ADP-glucose pyrophosphorylase mutant and its screening method and application. Background technique [0002] Starch plays an important role in human life. According to statistics, more than 80% of the world's starch comes from corn, and the proportion in the United States is as high as 95%. Among them, ADP-glucose pyrophosphorylase (ADP-Glucosepyrophosphorylase, abbreviated as AGPase) is a key enzyme in the process of plant starch synthesis. ADPG, which catalyzes the generation of 1-P-Glucose and ATP, participates in the synthesis of starch as the substrate of starch synthase. Its activity is a key factor in determining the rate of starch accumulation. A gene mutation encoding a subunit of AGPase in Arabidopsis leaves can reduce the enzyme activity to 7%, and starch accumulation to 26% (Lin T P, Caspar T, Somenrllle C. Starch deficient mutant Arabidopsis with low ADP-glucose pyrophosphorylase activity lacks one of the sub...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/12C12N15/82C12N15/29A01H5/00A01H6/46A01H6/00
CPCC12N9/1241C12N15/8202C12N15/8261C12Y207/07027
Inventor 王章英房伯平陈新亮陈景益张雄坚黄立飞罗忠霞
Owner CROP RES INST GUANGDONG ACAD OF AGRI SCI
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