ADP-glucose pyrophosphorylase mutant and screening method and application thereof

A phosphorylase mutant, glucose coke technology, applied in biochemical equipment and methods, applications, botanical equipment and methods, etc., can solve the problem of insufficient attention to research on high starch varieties

Active Publication Date: 2015-11-25
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

Method used

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  • ADP-glucose pyrophosphorylase mutant and screening method and application thereof
  • ADP-glucose pyrophosphorylase mutant and screening method and application thereof
  • ADP-glucose pyrophosphorylase mutant and screening method and application thereof

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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 NdeI is added at the 'end, and the recognition site of restriction endonuclease KpnI is added at the 3' end; SacI recognition site of endonuclease, the primer sequence is as follows:

[0083] The primer pair for amplification of the large subunit Shrunken-2 cDNA of maize endosperm AGPase is as follows:

...

Embodiment 1b

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

[0138] The AGPase large subunit Shrunken-2 cDNA was double-digested with restriction endonucleases NdeI and KpnI, and then connected to the prokaryotic expression vector that had been double-digested with the same enzymes to obtain a recombinant vector containing the AGPase large subunit Shrunken-2 cDNA; Sweet potato small subunit SIcDNA was double-digested with restriction endonucleases NcoI and SacI, and then ligated with the recombinant vector of the above-mentioned AGPase large subunit Shrunken-2 at a molar ratio of 1:1 to obtain the recombinant cDNA containing the large and small AGPase subunit Gene prokaryotic expression vector, referred to as SSI;

[0139] The AGPase large subunit Shrunken-2 cDNA was double-digested with restriction endonucleases NdeI and KpnI, and then connected to the prokaryotic expression vector that had been double-digested with the same enzyme...

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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% (LinTP, CasparT, SomenrllleC. ). Maize endosperm shrunken-2 and brittle-2 mutants (respectively the deletion mutants of the large and sma...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12N15/82C12N15/29A01H5/00
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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