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Tripterygium wilfordii cryptomeridiol synthetase as well as coding gene and application thereof in biosynthesis

A technology of cedardiol and synthase, which is applied in the field of synthetic biology of medicinal components and can solve problems such as tediousness

Active Publication Date: 2018-07-27
CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the use of multiple synthetic steps is cumbersome compared to the enzymes of the cederanediol biosynthetic pathway in plants

Method used

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  • Tripterygium wilfordii cryptomeridiol synthetase as well as coding gene and application thereof in biosynthesis
  • Tripterygium wilfordii cryptomeridiol synthetase as well as coding gene and application thereof in biosynthesis
  • Tripterygium wilfordii cryptomeridiol synthetase as well as coding gene and application thereof in biosynthesis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Tripterygium wilfordii suspension cell total RNA extraction and purification

[0045] Using improved CTAB method (CTAB Buffer: 2% CTAB (W / V); 100mmol L -1 Tris-HCl (pH 8.0); 25mmol L -1 EDTA; 2.0mol L -1 NaCl; 0.5g L -1 spermidine) to extract the total RNA of Tripterygium wilfordii suspension cells. The extracted RNA was purified using an RNA purification kit (Tiangen Biochemical Technology Co., Ltd.).

Embodiment 2

[0046] Example 2 TwCS gene full-length cDNA clone

[0047] 1. Primer Design

[0048] The full-length sequence fragment of the gene was screened according to the transcriptome data annotation of Tripterygium wilfordii, and 5'RACE and 3'RACE primers were designed. The primer sequences are as follows:

[0049] 5' RACE GTACCGTAAGCATCGTATGTGTCG (SEQ ID NO: 3)

[0050] 3' RACE CTATGAAGAGGACGAGTCTCGG (SEQ ID NO: 4)

[0051] 2. PCR amplification

[0052] Using PrimeScript 1 st The Strand cDNA Synthesis Kit (Takara Company) was used to reverse transcribe the RNA obtained in Example 1 into RACE Ready first-strand cDNA. Refer to SMARTer TM RACE kit instruction manual for rapid amplification of cDNA ends.

[0053] The 3' and 5' ends of the DNA sequence of SEQ ID No.1 were obtained by the RACE method, and then primers were designed according to the sequence information. The primer sequences were as follows:

[0054] TwCS-F ATGGCAGCGACCACCCCAATCCAC (SEQ ID NO: 5)

[0055] TwCS-RTT...

Embodiment 3

[0059] Example 3 plasmid construction

[0060] 1. Cloning of promoters and terminators

[0061] Both the yeast promoter and terminator used in this example are publicly available on the SGD website (https: / / www.yeastgenome.org).

[0062] The total DNA of yeast BY4741 was extracted using a yeast genome extraction kit (Tiangen Biochemical Technology Co., Ltd.). Then use this DNA as a template to design the following primers:

[0063] TEF1p-F ATAGCTTCAAAATGTTTCTACTC (SEQ ID NO: 7)

[0064] TEF1p-R TTTGTAATTAAAACTTAGATTAG (SEQ ID NO: 8)

[0065] FBA1t-F GTTAATTCAAATTAATTGATATAG (SEQ ID NO: 9)

[0066] FBA1t-R AGTAAGCTACTATGAAAGACTTT (SEQ ID NO: 10)

[0067] Promoter TEF1p (TEF1SGD ID: S000006284) and terminator FBA1t (FBA1SGD ID: S000001543) fragments were obtained by PCR amplification (the specific steps were the same as in Example 2).

[0068] Using the pYX212 plasmid as a template, the promoter TPIp and the terminator pYX212t were obtained by PCR amplification (the specif...

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Abstract

The invention relates to tripterygium wilfordii cryptomeridiol synthetase as well as a coding gene and applications of the tripterygium wilfordii cryptomeridiol synthetase and the coding gene in plantbreeding and biosynthesis. A cDNA full-length sequence of the novel tripterygium wilfordii cryptomeridiol synthetase gene is obtained through a polymerase chain reaction by conducting cloning for thefirst time; and then, an engineered yeast comprising the tripterygium wilfordii cryptomeridiol synthetase gene provided by the invention is constructed via synthetic biological means, so that the production of tripterygium wilfordii cryptomeridiol in the yeast is achieved.

Description

technical field [0001] The invention relates to a cederediol synthase and its coding gene, and the application of the cedardiol synthase and the coding gene in plant breeding and biosynthesis, and belongs to the field of synthetic biology of medicinal ingredients. [0002] The full-length cDNA sequence of the new cedardiol synthase gene (TwCS) in Tripterygium wilfordii Hook.F. was cloned for the first time by polymerase chain reaction, and then synthetic biology methods were used to construct yeast engineering bacteria. Yeast production of cryptodiol. Background technique [0003] Sesquiterpenoids are a class of natural compounds with various physiological and ecological functions, such as antimicrobial, defense against insects and herbivores, etc. At present, a large number of sesquiterpenoids have been found in both higher plants and lower plants, and are widely used in the fields of industry, agriculture and medicine. The biosynthesis of sesquiterpenes comes only from t...

Claims

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Application Information

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IPC IPC(8): C12N9/90C12N15/61C12N15/81C12N1/19C12N5/10A01H5/00
CPCC12N9/0006C12N9/90C12N15/81C12N15/8243C12Y101/01034C12Y504/99
Inventor 高伟黄璐琦童宇茹苏平关红雨
Owner CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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