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Resveratrol synthase gene RS derived from tetrastigma hemsleyanum diels et gilg and application thereof

A technology of resveratrol synthase and resveratrol, which is applied in the field of genetic engineering and can solve the problems of relatively little research at the molecular level

Active Publication Date: 2021-02-26
杭州鑫旺途科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on Clover mainly focuses on cultivation, seedling cultivation, extraction and separation of chemical components and pharmacological effects, etc., and there are few studies on its molecular level.

Method used

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  • Resveratrol synthase gene RS derived from tetrastigma hemsleyanum diels et gilg and application thereof
  • Resveratrol synthase gene RS derived from tetrastigma hemsleyanum diels et gilg and application thereof
  • Resveratrol synthase gene RS derived from tetrastigma hemsleyanum diels et gilg and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1 3

[0025] Example 1 Acquisition of the full-length sequence of the ThRS gene cDNA of Clover

[0026] The leaves of fresh plants of Clover were taken, wrapped in tin foil, and quickly frozen with liquid nitrogen, total RNA was extracted, and reverse transcribed into cDNA. Total RNA was extracted according to the instructions of TIANGEN RNAprep Pure Plant Total RNA Extraction Kit (DP441), and its integrity and concentration were detected by 1.0% agarose gel electrophoresis and a nucleic acid concentration detector.

[0027] Reverse transcription of total RNA according to Takara PrimeScript TM II 1st Strand cDNA Synthesis Kit instructions.

[0028] According to the existing transcriptome data and the RS gene sequence of the same family and genus in NCBI, BLAST analysis was carried out, and the sequence with the highest similarity was selected as the target gene sequence, and several pairs of primers were designed with the open reading frame sequence of this sequence as a template. ...

Embodiment 2

[0033] Example 2 Secondary structure and tertiary structure prediction and phylogenetic tree analysis of ThRS

[0034] Using the online software SOPMA (https: / / npsa-prabi.ibcp.fr / cgi-bin / npsa_automat.pl?page=nps a_sopma.html) to predict the secondary structure of ThRS protein in the resveratrol biosynthetic pathway, the results As shown in Figure 2, the protein consists of 45.93% α-helix (Alpha helix), 15.99% extended chain (Extended strand d), 6.40% β-turn (Beta turn) and 31.69% random coil (Random coil ), indicating that the α-helical structure is the backbone of the secondary structure of the protein RS.

[0035] Use the online software SWISS-MODEL (http: / / swissmodel.expasy.org / ) to predict the three-dimensional structure of the ThRS protein in the resveratrol biosynthesis pathway. The method used is X-ray, and the resolution is The result is as image 3 shown. The template number used is 3tsy.1.A, the sequence identity (Seq Identity) is 92.73%, the state of the oligonucl...

Embodiment 3

[0039] Example 3 Functional verification of the ThrRS gene

[0040] The cDNA sequence of the ThRS gene and the distribution of restriction sites on the plasmid vector pCMBIA1301 were analyzed, and PCR primers with SmaI and XbaI restriction sites were designed (upstream primer: TCCCCCGGGATGACTGAGTTGAAGAAG; downstream primer: GCTCTAGATTAAGTTGAGATTGTAGG) for constructing overexpression vector.

[0041] Clover cDNA was used as a template for PCR amplification, and the reaction system was the same as above. After the amplified product was subjected to 1.0% agarose gel electrophoresis, a kit was used to purify and recover the DNA fragment consistent with the target gene. The purified recovered product and the plasmid pCMBIA1301 were subjected to double enzyme digestion at 37°C, followed by agarose gel electrophoresis and then purified and recovered. The purified and recovered digested products were ligated with T4 DNA ligase and incubated overnight at 16°C. The ligation system was:...

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Abstract

The invention discloses a resveratrol synthase gene RS derived from tetrastigma hemsleyanum diels et gilg and an application of the resveratrol synthase gene RS. The nucleotide sequence of the resveratrol synthase gene RS is shown as SEQ ID NO. 1. According to the invention, tetrastigma hemsleyanum diels et gilg is used as a biological source to design a primer to amplify the cDNA sequence to obtain the resveratrol synthase gene RS, and the gene is used as one of key enzymes in a phenylpropane metabolic pathway and can be used for producing resveratrol.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, and mainly relates to a resveratrol synthase gene RS derived from cloverleaf and its application. Background technique [0002] In recent years, the country has attached great importance to traditional Chinese medicine and the government's strong support has driven the rapid development of the Chinese medicine industry and expanded the market demand for Tetrastigma hemsleyanum Diels et Gilg. The continuous deepening of research on the anti-tumor effect of cloverleaf has led to a rapid increase in the market price of cloverleaf in recent years, and its market demand is also increasing. [0003] In recent years, the gene regulation of the synthesis of medicinal plant secondary metabolites has become a very active frontier research field in molecular biology. The amount and composition of metabolites mainly depend on the key enzymes of biosynthesis and the expression levels in cells. At...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/10C12P7/22C12N15/70C12N1/21C12R1/19
CPCC12N9/1029C12Y203/01095C12N15/70C12P7/22
Inventor 夏鹏国张宇胡婉莹郑宇婕侯卓妮梁宗锁
Owner 杭州鑫旺途科技有限公司
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