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A group of glycosyl transferase, and applications thereof

A technology of glycosyltransferase and glycosyl transfer, which is applied in the fields of biotechnology and plant biology, and can solve the problems of cumbersome operation, large loss of raw materials, and increased cost.

Inactive Publication Date: 2015-12-23
周志华
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the large loss of raw materials in the chemical preparation method, the operation is cumbersome, and there are many by-products, resulting in increased costs and difficulty in increasing the yield
In addition, since the acquisition of total ginsenosides depends on the cultivation of ginseng, the market price of rare ginsenoside monomers produced by traditional methods is high
[0006] At present, there is still a lack of an effective method for producing rare ginsenosides Rg3, Rf and Rg2 in this field, so it is urgent to develop a variety of specific and efficient glycosyltransferases

Method used

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  • A group of glycosyl transferase, and applications thereof
  • A group of glycosyl transferase, and applications thereof
  • A group of glycosyl transferase, and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0229] Embodiment 1 Separation of glycosyltransferase and its coding gene

[0230] More than 100 cDNA sequences predicted to be glycosyltransferases were extracted from the published expression profile data of Panax genus plants, from which 60 full-length cDNA sequences were cloned and their expression and transglycosylation reactions were analyzed, among which 17 were The expression product has transglycosylation activity to ginsenogenin and saponin.

[0231]Ginseng RNA was extracted and reverse-transcribed to obtain ginseng cDNA. Carry out PCR amplification with this cDNA as template, use wherein primer pair 1 (SEQIDNO.:7,8); Primer pair 2 (SEQIDNO.:9,10); Primer pair 3 (SEQIDNO.:11,12); Primer pair 5 (SEQIDNO.:34,35); Primer pair 7 (SEQIDNO.:46,47); Primer pair 8 (SEQIDNO.:62,63); Primer pair 9 (SEQIDNO.:64,65) all obtain amplification product . As the DNA polymerase, the high-fidelity KOD DNA polymerase from Treasure Bioengineering Co., Ltd. was selected. PCR products ...

Embodiment 2

[0246] The construction of the yeast recombinant expression vector of embodiment 2 glycosyltransferase gene gGT29 and gGT29-3

[0247] The plasmids gGT29-pMD18T and gGT29-3-pMD18T constructed in Example 1 containing the gGT29 and gGT29-3 genes were respectively used as templates to amplify the target gene.

[0248] The forward primers used by gGT29 are all (SEQ ID NO.:36), and the KpnI recognition site is added to its 5' end: GGATCC; the reverse primers used are all (SEQ ID NO.:37), and the IIhoI recognition site is added to its 5' end: CTCGAG , the reverse primer was introduced into 6-HisTag for Western Blot detection, expression and purification.

[0249] The forward primers used in gGT29-3 are all (SEQ ID NO.:38), and the KpnI recognition site is added to its 5' end: GGATCC; the reverse primers used are all (SEQ ID NO.:39), and the IIhoI recognition site is added to its 5' end : CTCGAG, the reverse primer was introduced into 6-HisTag for Western Blot detection, expression ...

Embodiment 3

[0251] Example 3 Expression of Glycosyltransferase Genes gGT29 and gGT29-3 in Saccharomyces cerevisiae

[0252] The constructed expression plasmids gGT29-pYES2 and gGT29-3-pYES2 were transformed into Saccharomyces cerevisiae by electroporation, and spread on the screening plate SC-Ura (0.67% yeast-free basic nitrogen source of amino acids, 2% glucose) . Yeast recombinants were verified by colony PCR. The yeast recombinant colonies were picked and cultured in 10 mL of SC-Ura (2% glucose) medium at 200 rpm at 30° C. for 20 h. Collect the bacteria by centrifugation at 3500g at 4°C, wash the bacteria twice with sterile deionized water, resuspend the bacteria with induction medium SC-Ura (2% galactose), and inoculate into 50mL induction medium to make the OD 600 At about 0.4, the expression was induced at 30°C and 200rpm. Centrifuge at 3500g at 4°C to collect the cells induced to express for 12 hours, wash the cells twice with sterile deionized water, resuspend them in yeast lys...

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Abstract

The present invention relates to a group of glycosyl transferase and applications thereof, and specifically provides applications of the glycosyl transferase gGT29-7 and a derived polypeptide thereof in terpene compound glycosylation catalysis and new saponin synthesis, wherein the glycosyl transferase can specifically and efficiently transfer the glycosyl from a glycosyl donor to the first glycosyl on C-3 site and / or C-6 site of a tetracyclic triterpene compound so as to extend the sugar chain. The glycosyl transferase of the present invention can further be used for constructing artificial ly-synthesized rare ginsenosides and a variety of new ginsenosides and derivatives thereof.

Description

technical field [0001] The invention relates to the fields of biotechnology and plant biology, in particular, the invention relates to a group of new glycosyltransferases and their applications. Background technique [0002] Ginsenoside is a general term for the saponins isolated from ginseng and its congeners (such as Panax notoginseng, American ginseng, etc.), belonging to triterpenoid saponins, and is the main active ingredient in ginseng. At present, at least 60 saponins have been isolated from ginseng, some of which have been proven to have a wide range of physiological functions and medicinal value: including anti-tumor, immune regulation, anti-fatigue, heart protection, liver protection and other functions. [0003] Ginsenosides Rg3, Rf and Rg2 are all rare ginsenosides, which have very powerful above-mentioned physiological activities respectively. For example, ginsenoside Rg3 has good antitumor activity, can induce tumor cell apoptosis, and inhibit tumor cell metas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P33/00C12N9/10C12N15/54C12N5/10A01H5/00
CPCA01H4/00C07K14/415C12P19/18C12P33/00C12N9/10C12N15/63
Inventor 周志华严兴王平平魏勇军魏维许云鹏李晓东杨成帅
Owner 周志华
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