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The cdna sequence of the coding gene gl22395 of Chizhi terpene synthase and its application

A technology that encodes genes and terpene synthases, applied in the field of genetic engineering, can solve problems such as difficulties in extraction and separation, and unclear molecular mechanisms

Active Publication Date: 2018-04-10
INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The content of these three terpenes in Ganoderma lucidum is very small, and the extraction and separation are difficult, and the chemical synthesis is also facing great challenges
Isotope labeling experiments show that Ganoderma lucidum terpenoids are synthesized through the Mevalonic Acid Pathway (MVP), and lanosterol is a common cyclic precursor of terpenoids and ergosterol (an important component of fungal cell membranes). The research on the specific downstream synthesis pathway of Ganoderma lucidum terpenoids is still blank, and the molecular mechanism for the formation of a wide variety of Ganoderma lucidum terpenoids from a single precursor lanosterol is still unclear

Method used

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  • The cdna sequence of the coding gene gl22395 of Chizhi terpene synthase and its application
  • The cdna sequence of the coding gene gl22395 of Chizhi terpene synthase and its application
  • The cdna sequence of the coding gene gl22395 of Chizhi terpene synthase and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Obtaining the cDNA sequence of Ganoderma lucidum terpene synthase GL22395 encoding gene

[0035] 1. Materials and reagents

[0036] 1.1 Experimental materials

[0037] The Ganoderma lucidum strain used-Ganoderma lucidum was purchased from the China Common Microbial Culture Collection and Management Center, and the total RNA was extracted from Ganoderma haploid mycelium obtained by tissue culture.

[0038] 1.2 Enzymes and chemical reagents

[0039] DNA polymerase, dNTP Mixture, recombinant DNase I (RNase-free), DL5000DNAMaker, DL2000DNA Maker were purchased from TaKaRa; 2×EasyTaq PCR SuperMix was purchased from Beijing TransGen Biotechnology Co., Ltd; Trgptone (OXOID, England), yeast extract ( OXOID, England), vitamin B1 (Sigma), X-gal (Sigma), IPTG (Merck); other required drugs are all domestically pure.

[0040] 1.3 Kit

[0041] Total RNA extraction kit, PCR purification and gel extraction kit were purchased from QiaGen; 1st Strand cDNA synthesis kit and DNA A-Tailing ...

Embodiment 2

[0140] Example 2 Tissue expression analysis of GL22395 gene cDNA sequence

[0141] Extract the total RNA of mycelium, primordium and fruiting body of Ganoderma lucidum at three different growth stages, and use the reverse transcription kit (PROMEGA) for reverse transcription. Use the protein phosphatase 2A (PP2A) gene as the internal reference gene for fluorescence quantitative PCR analysis. The forward primer is: 5'-CTCGCACGCTACAACAAAG-3', and the reverse primer is: 5'-AGTCGGAAGTGGGTGGG-3'. The results show that the expression of this gene in mycelium is significantly higher than its expression in the primordium and fruiting body stage ( figure 1 ).

Embodiment 3

[0142] Example 3 Prokaryotic expression of GL22395 gene cDNA sequence

[0143] 1. According to the full-length cDNA sequence of Ganoderma lucidum terpene synthase, primers for PCR amplification of a complete open reading frame were designed, and restriction enzyme cleavage sites BamHI and Xhol I were added to the forward and reverse primers respectively. The designed primers are:

[0144] Forward primer: 5′-CGCGGATCCATGAGCGTC-3′

[0145] Reverse primer: 5'-CCGCTCGAGTCAGATCGA-3'

[0146] Using the full-length cDNA fragment as a template, after PCR amplification, ensure that the reading frame of the terpene synthase gene of Ganoderma lucidum is completely correct, and use BamHI and XholⅠ endonuclease for digestion reaction to recover the target fragment 1260bp; E. coli expression vector pET28a BamHI and XholI endonuclease digestion reaction was performed to recover the target fragment 5kb. The target fragment of the digested Ganoderma terpene synthase gene was cloned into the digested...

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Abstract

The invention relates to a cDNA (complementary deoxyribonucleic acid) sequence of a ganoderma lucidum terpene synthase GL22395 encoding gene and an application of the cDNA sequence. The cDNA sequence of the ganoderma lucidum terpene synthase GL22395 encoding gene is represented as Seq ID No.2, and 419 amino acids are encoded. The ganoderma lucidum terpene synthase gene GL22395 is adopted to transform escherichia coli, and endogenous FPP (farnesyl pyrophosphate) is taken as a substrate, so that terpenoid is synthesized in an internal heterogeneous source of escherichia coli.

Description

Technical field [0001] The invention belongs to the field of genetic engineering, and specifically relates to the cDNA sequence of the ganoderma terpene synthase GL22395 encoding gene and its application. Background technique [0002] Ganoderma is the general name of Basidiomycetes, Polyproraceae, Ganoderma fungi, Ganoderma lucidum and G. sinense. As a traditional medicinal fungus in my country, Ganoderma lucidum has been used in disease treatment and health care for thousands of years and has a long history of medicinal use. The medicinal records of Ganoderma lucidum first appeared in the "Shen Nong Materia Medica" more than two thousand years ago. At present, Ganoderma lucidum has been included in the American Herbal Pharmacopoeia and Therapeutic Compendium, and its medicinal value has been obtained. Widely recognized by countries all over the world. Ganoderma contains a variety of biologically active substances. So far, more than 400 different compounds have been identified,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/63C12N1/21C12N15/11C12N15/113
CPCC12N9/88C12N15/11C12Y402/03047
Inventor 孙超陈士林曾欣宜王丽芝
Owner INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI
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