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Gene encoding phosphoglucomutase in kelp, its protein and application

A technology of glucose phosphate and mutase, applied in the fields of genetic engineering and biology, can solve the problems of low catalytic activity, poor metal ion tolerance, and reduced activity, and achieve the effect of ensuring applicability

Active Publication Date: 2020-06-02
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The PGMs that have undergone performance research mainly have the following defects: (1) the catalytic activity of most PGMs is relatively low, and cannot be used in industrial production at all, such as the PGMs disclosed in documents 1 and 2; (2) some catalytic activities are still low. Possible PGMs have relatively poor tolerance to metal ions, such as the PGMs disclosed in Document 3; (3) the optimum reaction temperature of some active PGMs is too extreme, such as those disclosed in Documents 2, 4 and 5 PGM, its optimum reaction temperature is even as high as 70°C and 90°C, while the normal temperature of organisms is around 30-37°C, and plants will die under too high a temperature, which makes these PGMs in plants The activity is obviously reduced at normal temperature, so it is impossible to play the catalytic activity normally in plants
Unfortunately, there are currently no such PGM resources reported

Method used

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  • Gene encoding phosphoglucomutase in kelp, its protein and application
  • Gene encoding phosphoglucomutase in kelp, its protein and application
  • Gene encoding phosphoglucomutase in kelp, its protein and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Cloning expression and isolation and purification of SjaPGM2 gene in embodiment 1 Laminaria

[0040] 1. Obtain a PGM gene named SjaPGM2 gene from the kelp embryospore gene database, and the nucleotide sequence of the SjaPGM2 gene is the sequence shown in SEQ ID NO:1. The full sequence of the SjaPGM2 gene was synthesized (Shanghai Xuguan Biotechnology Development Co., Ltd. was commissioned to synthesize), and at the same time, an EcoRI restriction site was introduced at its 5' end, and a NotI restriction site and corresponding protection bases were introduced at its 3' end to obtain SjaPGM2 gene sequence.

[0041] 2. Ligate the SjaPGM2 gene synthesized in step 1 between the EcoRI and NotI sites of the pET32a vector (purchased from Novagen) to obtain the pET32a-SjaPGM2D recombinant vector; transform the obtained pET32a-SjaPGM2 recombinant vector into Escherichia coli E.coli BL21(DE3) competent cells (purchased from Takara Company), spread on LB solid culture plates conta...

Embodiment 2

[0042] Identification of embodiment 2 recombinant protein

[0043] SDS polyacrylamide gel electrophoresis is carried out to the SjaPGM2 protein in embodiment 1 and the bacterium liquid before pure flower, and polyacrylamide gel electrophoresis figure is as follows figure 1 As shown, the molecular weight of SjaPGM2 protein is about 85KD, which is in line with the expected size.

Embodiment 3

[0044] Example 3 Functional verification of phosphoglucomutase and determination of enzyme activity parameters

[0045] Enzyme activity assay method: the volume of the reaction system is 200 μl, according to the reaction system in Table 1, sequentially add reagents other than the recombinant protein SjaPGM2 protein obtained in Example 1, mix well and place in a metal bath at 30°C for 2 minutes; after incubation, place Measure the absorbance value at 340nm under the ultraviolet spectrophotometer; then add the substrate glucose-6-phosphate to initiate the reaction, and start timing, and measure the changes in the absorbance value at 340nm at 0min, 6min and 12min; The corresponding amount of NADP reduced to NADPH was used to calculate the enzyme activity.

[0046] Table 1 SjaPGM2 protein enzyme activity detection reaction system

[0047]

[0048] Determination results: Compared with the blank control (that is, reacting without adding recombinant protein SjaPGM2 protein in the...

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Abstract

The invention belongs to the biotechnical field, particularly belongs to the field of genetic engineering, and particularly relates to a gene for encoding phosphoglucomutase in kelp, a protein and applications of the gene. The encoded gene is named as SjaPGM2 gene, the sequence of which is shown as SEQ ID NO:1, the encoded protein is named as SjaPGM2 protein, the sequence of which is shown as SEQ ID NO:2, and is a PGM. The SjaPGM2 protein also has higher catalytic activity compared with the prior art, more excellent metal ion stability and thermal stability, milder optimal reaction temperature, higher catalytic efficiency and more applicable to improvement of plant nutrition quality compared with the prior art, thus providing a very suitable gene and protein resources for the genetic engineering application of PGM.

Description

technical field [0001] The invention belongs to the field of biotechnology, specifically to the field of genetic engineering, and more specifically relates to a gene encoding phosphoglucomutase in sea-tangle, its protein and its application. Background technique [0002] Phosphoglucomutase gene (PGM) is a gene ubiquitous in organisms, ubiquitously present in plants, animals and microorganisms, the main biological effect of its encoded phosphoglucomutase (Phosphoglucomutase, PGM) is Participate in catalyzing the reversible conversion between glucose-1-phosphate (G-1-P) and glucose-6-phosphate (G-6-P), which is closely related to the glucose metabolism of organisms. This reaction mechanism includes two phosphate group transfer reactions: first, the phosphate group is transferred from the serine in the active site of phosphoglucomutase to the substrate to generate 1,6-diphosphate glucose (Glucose-1,6-P) intermediate. Then the phosphate group is transferred back to the phospho...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/61C12N9/90
CPCC12N9/90C12Y504/02002
Inventor 刘涛池姗刘翠
Owner OCEAN UNIV OF CHINA
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