Corn embryosperm ADP- glucose pyrophosphorylase mutant and its screening method and application

A technology of phosphorylase mutants and corn endosperm, applied in biochemical equipment and methods, applications, botanical equipment and methods, etc., can solve the problems of no discovery, achieve the effects of enhanced affinity and broad application prospects

Inactive Publication Date: 2007-05-30
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In maize kernels, the changes in the content of the large and small subunits of AGPase are consistent with the transcription levels of its coding genes Shrunken-2 and Brittle-2, and no other level of regulation has been found so far; in te

Method used

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  • Corn embryosperm ADP- glucose pyrophosphorylase mutant and its screening method and application
  • Corn embryosperm ADP- glucose pyrophosphorylase mutant and its screening method and application

Examples

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Embodiment 1

[0049] Example 1. Obtaining of Maize Endosperm ADP-Glucose Pyrophosphorylase Mutant and Its Encoding Gene

[0050] 1. Obtaining the large and small subunit cDNA of maize endosperm AGPase

[0051] 1. Primer design

[0052] Two pairs of RT-PCR primers (Sh and Bt) were designed according to the cDNA sequences of the corn endosperm AGPase large subunit gene Shrunken-2 and small subunit gene Brittle-2 (GenBank numbers: M81603, AF334959), and for the convenience of vector construction , when designing the primers, a special design was made for the following two points: a. The recognition site of the restriction endonuclease NdeI was added at the 5' end of Shrunken-2, and the recognition site of the restriction endonuclease SmaI and SacI was added at the 3' end of Shrunken-2 site; b. Because there is a restriction endonuclease NcoI recognition site at the ATG position at the 5' end of Brittle-2, the A at position 39 at the 5' end of Brittle-2 is mutated to C, and the presence at thi...

Embodiment 2

[0085] Example 2, Determination of Enzyme Activity and Glycogen Content of Corn Endosperm AGPase Mutant

[0086] 1. Determination of AGPase activity in crude extract of corn endosperm AGPase mutant

[0087] 1. Obtaining crude extract of corn endosperm AGPase mutant

[0088] Pick the Escherichia coli glgC mutant strain (control) containing the wild-type corn endosperm AGPase gene and the single colonies of the mutant strains 10-3 and 10-3-53 obtained through screening in Example 1, and inoculate them in LB liquid medium (containing 100 μg / mL ampicillin), cultured at 37°C for 12-24 hours, and then transferred the overnight cultured bacterial solution to fresh LB liquid medium (containing 100 μg / mL ampicillin) at a ratio of 1:100. Shake culture at 37°C and 200rpm for 3-4 hours to OD 600 After the concentration is 0.6-0.8, add a final concentration of 0.1mM IPTG and induce at room temperature for 12-24 hours. After the cultivation, the bacteria were collected by centrifugation ...

Embodiment 3

[0095] Embodiment 3, the purification of corn endosperm AGPase mutant

[0096] 1. Protein Solubility Analysis

[0097] First, the bacterium liquid of the corn endosperm AGPase mutant strain 10-3-53 obtained in Example 2 induced by IPTG for 12-24 hours was centrifuged at 4° C. and 5000 rpm for 5 minutes to collect the induced cells, discard the supernatant, and use 1 / 10 volumes of TE (50mM Tris-HCl pH 8.0, 0.2mM EDTA) to resuspend the cell pellet, then add 10mg / mL lysozyme (prepared with fresh TE buffer) to a final concentration of 100μg / mL, then add 1 / 10 volume of Triton-100, incubate at 30°C for 15 minutes, put the centrifuge tube in an ice-water bath, ultrasonicate, then centrifuge at 12,000rpm at 4°C for 15 minutes, take the supernatant and precipitate for SDS-PAGE electrophoresis: supernatant Contains soluble protein, you can add an equal volume of 2×SDS loading buffer, boil for 5 minutes, centrifuge at 12000rpm for 5 minutes at room temperature, take 30mL supernatant fo...

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Abstract

The invention discloses a maize-endosperm ADP-glucose pyrophosphorylas mutant and sieving method and application, which comprises the following steps: 1) mutating cDNA of large and small subunit of maize-endosperm AGPase through alcaine hydroxylamine mutation agent randomly; 2) transmitting mutated cDNA and wild-type cDNA into escherichia coli glgC mutation germ; sieving positive monoclonal; 3) seeding positive monoclonal line on the Conberg rich solid board to culture under 35-39 deg.c; 4) dyeing; comparing the color; obtaining deep color mutation as maize-endosperm ADP-glucose pyrophosphorylas mutant.

Description

technical field [0001] The present invention relates to plant ADP-glucose pyrophosphorylase mutants and their screening methods and applications, in particular to ADP-glucose pyrophosphorylase mutants derived from corn endosperm and their screening methods and the mutants and their coding genes Application in corn variety improvement. Background technique [0002] Starch plays an important role in human life, so researchers have not stopped studying starch synthesis. In the fields of agriculture and forestry, people have long been committed to increasing the yield of crops (such as corn, etc.) to provide enough food and industrial raw materials for the continuously growing world population. At the same time, from the perspective of sustainable development and environmental protection, the demand for the yield and quality of corn production will increase and improve day by day. In addition, because plant cells or organs with a high starch content absorb less fat or frying o...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12N15/54C12N15/82
Inventor 王国英王章英陈小平王建华
Owner CHINA AGRI UNIV
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