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Isopentenyl pyrophosphate isomerase gene and its application

A technology of isopentenyl pyrophosphate and isomerase, applied in the field of genetic engineering, can solve the problem of low efficiency of terpenoids

Active Publication Date: 2020-03-31
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The present invention aims to solve the technical problem that the efficiency of synthetic terpenoids by engineering bacteria is not high, and provides a kind of engineering bacteria with strong synthesis pathway of terpenoids and its application.

Method used

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  • Isopentenyl pyrophosphate isomerase gene and its application
  • Isopentenyl pyrophosphate isomerase gene and its application
  • Isopentenyl pyrophosphate isomerase gene and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1: Obtaining of gene fragments

[0026] 1. Extraction of total RNA from poplar leaves

[0027] Collect poplar leaves, use RNeasy Plant Mini Kit (Qiagen company) to extract poplar leaf total RNA, carry out according to the kit instruction method, carry out electrophoresis ( figure 1 ) to verify the quality of RNA extraction, it can be seen that the integrity of the RNA is good, and subsequent experiments can be performed.

[0028] 2. Preparation of RACE-Ready cDNA

[0029] The reverse transcription system for the first strand of RACE-Ready cDNA is as follows:

[0030] The method for obtaining full-length cDNA is SMARTer-RACE, using PCR cDNA Synthesis Kit (Clontech Company) was carried out, and the primers and reagents used below were all except GSP Provided in the PCR cDNA SynthesisKit, follow the kit instructions.

[0031] The reverse transcription system for the first strand of RACE-Ready cDNA is as follows:

[0032]

[0033] 3. Design of gene-spe...

Embodiment 2

[0037] Example 2: Obtaining the full length of the Paidi gene coding region

[0038] 1. Obtaining the end sequence of 3'-RACE cDNA

[0039] Using 3'-RACE-Ready cDNA of Populus alba as template, UPM and GSP as primers for amplification

[0040] reaction system:

[0041]

[0042] Reaction conditions:

[0043]

[0044] 3'-RACE agarose gel detection showed a single bright Populus alba DNA amplified band, connected to the T vector, transformed competent cells, selected positive clones for Sanger sequencing, and obtained the 3' end cDNA sequence.

[0045] 2. Obtaining the end sequence of 5'-RACE cDNA

[0046] The 5'-RACE-Ready cDNA of Populus alba was used as a template, and UPM and GSP were used as primers for amplification.

[0047] reaction system:

[0048]

[0049] Reaction conditions:

[0050]

[0051] A single bright amplified band was detected by 5'-RACE agarose gel, and one of them was selected to be connected to the T vector, transformed into competent cells...

Embodiment 3

[0054] Embodiment 3: Construction of Escherichia coli isoprene production strain

[0055] 1. Construction of Escherichia coli expression vector p2-0

[0056] The full-length primer sequences are as follows:

[0057]

[0058] Using MB-F and MB-R as primers and the p2 plasmid as a template, the obtained gene fragment MB was double digested with SalI and PstI (TAKARA company), and the pSB1c expression vector (constructed in our laboratory, arabinose-inducible promoter , the sequence shown in SEQ ID No.3) was double digested with XhoI and PstI, the MB gene fragment was connected to the pSB1c vector to obtain p2-0, transformed into trans5α competent cells, and positive clones were selected for sequencing, p2-0 The nucleotide sequence of is SEQ ID No.4.

[0059] 2. Construction of Escherichia coli expression vector p2-paidi

[0060] The sequences of the full-length primers PAFa and PARa are as follows:

[0061] PAFa: 5'ATCGGctgcagATGGGTGACGCTCCTGATGC 3'

[0062] PARa: 5'ATCC...

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Abstract

The invention relates to an isopentenyl diphosphate isomerase gene and applications thereof. A technical problem that efficiency of synthesis of terpenoids using engineering bacteria is low is solved. The invention provides the isopentenyl diphosphate isomerase gene, proteins obtained via expression of the isopentenyl diphosphate isomerase gene, and preparation method and applications of a prokaryotic expression vector containing the isopentenyl diphosphate isomerase gene and engineering bacteria capable of producing terpenoids. The isopentenyl diphosphate isomerase gene can be widely applied in the field of terpenoid preparation.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an isopentenyl pyrophosphate isomerase gene and its application. Background technique [0002] Terpenoids are compounds with isoprene structural units (C 5 h 8 )n compounds of the general formula and their derivatives containing oxygen and different degrees of saturation, terpenoids are obtained through their precursor isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate ( DMAPP) condensation. [0003] Terpenoids exist widely in nature, and higher plants, fungi, microorganisms, insects and marine organisms can synthesize terpenoids. Lycopene, astaxanthin, carotene, vitamin A, coenzyme Q10, paclitaxel and artemisinin are all terpenoids. Terpenoids have many physiological activities, such as expectorant, cough, carminative, sweating, insect repellent, analgesic, etc. They are a kind of relatively important active ingredients in Chinese herbal m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/61C12N9/90C12N15/70C12N1/21C12P5/02
Inventor 李春胡开蕾韩莉张莎莎赖小勤刘伟丰徐欣怡高珊陶勇
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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