Method used for synthesizing plurality of sesquiterpenoid compounds using salviae miltiorrhizae SmTPS3 gene

A sesquiterpene and compound technology, applied in the field of identification and functional verification, terpenoid synthase gene cloning, can solve the problem of unresolved volatile substances

Active Publication Date: 2018-11-13
INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The biosynthetic pathways of these volatile substances are still not resolved

Method used

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  • Method used for synthesizing plurality of sesquiterpenoid compounds using salviae miltiorrhizae SmTPS3 gene
  • Method used for synthesizing plurality of sesquiterpenoid compounds using salviae miltiorrhizae SmTPS3 gene
  • Method used for synthesizing plurality of sesquiterpenoid compounds using salviae miltiorrhizae SmTPS3 gene

Examples

Experimental program
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Effect test

Embodiment 1

[0017] Example 1 Cloning and structural analysis of SmTPS3 gene

[0018] 1) Using RT-qPCR technology to predict the differential expression of SmTPS3 gene in different tissues and organs of Salvia miltiorrhiza. The results are presented in the form of a histogram, such as figure 2 Shown. It can be found that SmTPS3 is highly expressed in the stems of Salvia miltiorrhiza.

[0019] 2) Design primers based on the SmTPS3 sequence, and use Salvia miltiorrhiza cDNA as a template for amplification to obtain a nucleotide sequence of 1614 bp in length, such as SEQ ID No. 1. The amino acid sequence of Salvia miltiorrhiza SmTPS3 was obtained after translation according to the full-length cDNA sequence, as SEQ ID No.2.

Embodiment 2

[0020] Example 2 Construction of Prokaryotic Expression System of SmTPS3 Gene and Detection of Metabolites

[0021] 1) Select BamH I / Xho I as the restriction site, perform restriction digestion and ligation reaction on SmTPS3 and pET28a vector to construct the heterologous expression vector pET28a-SmTPS3 of the gene.

[0022] 2) Transform pET28a empty vector and pET28a-SmTPS3 into competent cells BL21 (DE3) respectively, and spread them on LB solid medium plates containing 50 mg / L Kana (kanamycin) to select positive clones. Single colonies of positive clones were selected to be inoculated into LB liquid medium containing corresponding antibiotics (50 mg / L Kana) and cultured overnight. On the next day, transfer and expand the culture at a ratio of 1:50. When the OD600 of the bacterial solution reaches between 0.4-0.6, add 0.5mM IPTG, in a shaker at 25℃, avoid light to induce protein expression for 20h, rotating speed Set to 110r / min.

[0023] 3) Take 10mL of the induced bacterial so...

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Abstract

The invention discloses a terpene synthases gene sequence (SmTPS3) mainly participating in synthesis of a plurality of salviae miltiorrhizae secondary metabolites sesquiterpenoids such as zingiberene.The SmTPS3 gene possesses a nucleotide sequence represented by SEQ ID No.1, and the protein encoded by the gene possesses an amino acid sequence represented by SEQ ID No.2. The SmTPS3 gene sequence is obtained based on salviae miltiorrhizae genome and transcriptome data, a pET28a-SmTPS3 prokaryotic expression vector is constructed, the catalysis products of SmTPS3 are detected in Escherichia coli, so that the gene functions of SmTPS3 are verified. It is shown by research results that: SmTPS3 is capable of catalyzing synthesis of a plurality of sesquiterpenoid components including zingiberene,alpha-bergamot oil alkene, beta- sesquiphellandrene; zingiberene possesses anti-virus activity, anti-fertility activity, and antiulcer activity, and can be taken as food flavoring agent and cosmeticraw material; bergamot essential oil possesses excellent curative effects in alleviating anxiety and depression. The method is capable of providing salviae miltiorrhizae terpene synthases gene synthesis of sesquiterpenoid compounds with foundation, and promoting medicine development and industry development of sesquiterpenoid compounds, such as zingiberene and alpha-bergamot oil alkene, based onSmTPS3 catalysis synthesis.

Description

Technical field [0001] The invention belongs to the technical fields of plant molecular biology and plant genetic engineering, and specifically relates to a method for cloning, identification and functional verification of a terpene synthase gene involved in the synthesis of gingerene and other sesquiterpene compounds in Salvia miltiorrhiza. Background technique [0002] Salvia miltiorrhiza Bunge is a medicinal plant of the Lamiaceae and Salvia genus. Salvia is a large branch of plants in the Lamiaceae. The plants in the genus are mostly aromatic and rich in volatile substances. At present, more than 700 chemical components have been isolated from 134 species of Salvia plants, mainly terpenoids, steroids and polyphenols. These volatile substances are mostly antiparasitic, antibacterial, antioxidant, Anti-inflammatory and other effects. As an important medicinal plant in the genus Salvia, Salvia miltiorrhiza contains a lot of volatile substances in its body. The biosynthetic pa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/60C12N9/88C12N15/70C12P5/00
CPCC12N9/88C12N15/70C12P5/002C12Y402/03
Inventor 罗红梅刘琬菁
Owner INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI
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