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

A phosphorylase mutant, glucose coke technology, applied in biochemical equipment and methods, applications, botanical equipment and methods, etc., can solve problems such as no discovery, and achieve the effect of enhanced affinity and broad application prospects.

Inactive Publication Date: 2009-11-25
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 terms of thermal stability, due to the two Each of the small subunits has cysteine ​​at the N-terminus, thus forming a relatively stable disulfide bond structure, while the large and small subunits of maize AGPase do not have this structure

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

Examples

Experimental program
Comparison scheme
Effect test

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 restriction endonuclease Nde I was added at the 5' end of Shrunken-2, and the restriction endonuclease Sma I and SacI were added at the 3' end b. Because there is a restriction endonuclease Nco I 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 The second Nco I recognition site exists at this site, and...

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 cells were collected by centrifugation at ...

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 corn endosperm ADP-glucose pyrophosphorylase mutant, a screening method and application thereof. The screening method comprises the following steps: 1) using hydroxylamine hydrochloride mutagen to randomly mutate the large and small subunit cDNA of corn endosperm AGPase under the condition of 70-80°C warm bath; The wild-type maize endosperm AGPase large and small subunit cDNAs were transformed into Escherichia coli glgC mutant strains, and positive single clones were screened; 3) The positive clones were streaked and inoculated on a Conberg enriched solid culture plate, and cultured at 35-39°C; 4) Use iodine-potassium iodide solution to stain the single colony grown on the Conberg enriched solid culture plate, and compare the color with the control colony. The mutant strain is darker than the control color. After sequencing verification, the corn endosperm ADP- Glucose pyrophosphorylase mutants. The maize endosperm ADP-glucose pyrophosphorylase mutant and its screening method of the present invention will play an important role in the improvement of maize varieties.

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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Patent Type & Authority Patents(China)
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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