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UGD gene in kelp, protein thereof and application

A protein and gene technology, applied in genetic engineering, plant gene improvement, application, etc., can solve problems such as lack of cloning, and achieve the effects of excellent gene resources and enzyme resources, high specific activity, and wide optimal reaction temperature range

Active Publication Date: 2017-10-24
青岛海大蓝科生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the cloning of UGD genes related to algin biosynthesis is very scarce at present, and there is only one GMD gene (EsGMD1) encoding GDP-mannose-6-dehydrogenase in brown algae that has been published, and there is no UGD gene.

Method used

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  • UGD gene in kelp, protein thereof and application
  • UGD gene in kelp, protein thereof and application
  • UGD gene in kelp, protein thereof and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The acquisition of UGD gene in the sea-tangle of embodiment 1

[0031]According to the whole genome sequence information of kelp (Sacchrina japonica), a UGD gene was obtained, which was named SjaUGD gene. The nucleotide sequence of the SjaUGD gene is the sequence shown in SEQ ID NO:1. Synthesize the entire sequence of the SjaUGD gene (Shanghai Xuguan Biotechnology Development Co., Ltd. was commissioned to synthesize by Sangon Bioengineering Co., Ltd.), and at the same time introduced EcoRI and NotI restriction sites and corresponding protective bases at both ends to obtain the SjaUGD gene sequence.

Embodiment 2

[0032] Cloning expression and separation and purification of embodiment 2 SjaUGD gene

[0033] The SjaUGD gene synthesized in Example 1 was connected between the EcoRI and NotI sites of the pET32a vector (purchased from Novagen) to obtain the pET32a-SjaUGD recombinant vector; the obtained pET32a-SjaUGD recombinant vector was transformed into Escherichia coli E.coli BL21 (DE3) Competent cells (purchased from Takara Company), spread on LB solid culture plates containing 100 μg / mL ampicillin, and cultivate overnight at 37°C; inoculate positive clones on LB solid culture plates containing 100 μg / mL ampicillin In the culture medium, cultivate at 37°C until the OD600 of the bacterial solution is 0.4, add a final concentration of 0.5mM IPTG, and induce the expression of the target protein at 25°C and 160rpm overnight; Centrifuge for 5 minutes, discard the supernatant, and place the tube upside down on absorbent paper; add 1 / 2 volume of the precipitate to 50mM PBS buffer (PH=7.5) pre-...

Embodiment 3

[0034] Identification of embodiment 3 recombinant protein

[0035] SDS polyacrylamide gel electrophoresis is carried out to the SjaUGD protein that embodiment 2 obtains, polyacrylamide gel electrophoresis figure is as follows figure 1 As shown, the molecular weight of SjaUGD protein is about 50KD, which is in line with the expected size.

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Abstract

The invention belongs to the technical field of biology, particularly belongs to the field of genetic engineering, and more particularly relates to UGD gene in kelp, protein thereof and application. the UGD gene is from the kelp and is named as SjaUGD gene, and a nucleotide sequence of the SjaUGD gene is a sequence shown as SEQ ID NO:1. The invention further provides GDP-mannose-6-dehydrogenase which is coded by the SjaUGD gene, an amino acid sequence of GDP-mannose-6-dehydrogenase is a sequence shown as SEQ ID NO:2 while specific activity of the same is 7.99nanokatals.mg-1, and GDP-mannose-6-dehydrogenase has wider optimum reaction temperature range and higher thermostability. Excellent gene resources and enzyme resources are provided for biological preparation of algin.

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 kelp UGD gene, its protein and its application. Background technique [0002] Alginate, also known as alginic acid polysaccharide, its sodium salt form is called sodium alginate (alginate), which is composed of β-D-mannuronic acid (β-D-mannuronic acid, referred to as M) and α-L-guro Uronic acid (α-L-guluronic acid, G for short) is a linear polysaccharide linked by 1,4 glycosidic bonds. So far, it can be considered that the biological sources of alginate are only Phaeophyta plants, such as kelp, sargassum, macroalgae, etc.; Azotobacter vinelandii). [0003] Alginate can be used as a food thickener, emulsifier, quality improver, etc.; in the pharmaceutical industry, alginate can be used as a substitute for blood plasma, hemostatic agent, scald gauze, dental impression agent, pharmaceutical excipient, and new barium m...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21C12P19/04C12R1/19
CPCC12N9/0006C12P19/04C12Y101/01022
Inventor 池姗刘涛刘翠
Owner 青岛海大蓝科生物科技有限公司
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