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CDNA (complementary deoxyribonucleic acid) sequence of ganoderma lucidum terpene synthase GL22395 encoding gene and application of cDNA sequence

A technology encoding gene and terpenoid synthase, applied in the field of genetic engineering, can solve the problems of unclear molecular mechanism, difficulty in extraction and separation, etc.

Active Publication Date: 2015-12-16
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 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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  • CDNA (complementary deoxyribonucleic acid) sequence of ganoderma lucidum terpene synthase GL22395 encoding gene and application of cDNA sequence
  • CDNA (complementary deoxyribonucleic acid) sequence of ganoderma lucidum terpene synthase GL22395 encoding gene and application of cDNA sequence
  • CDNA (complementary deoxyribonucleic acid) sequence of ganoderma lucidum terpene synthase GL22395 encoding gene and application of cDNA sequence

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Embodiment 1

[0034] Example 1 Acquisition of the cDNA sequence of the gene encoding Chizhi terpene synthase GL22395

[0035] 1. Materials and reagents

[0036] 1.1 Experimental materials

[0037] The ganoderma lucidum strain used—red lucidum was purchased from the China General Microorganism Culture Collection and Management Center, and total RNA was extracted from ganoderma haploid hyphae obtained from tissue culture.

[0038] 1.2 Enzymes and chemical reagents

[0039] DNA polymerase, dNTPMixture, recombinant DNaseI (RNase-free), DL5000DNAMaker, DL2000DNAMaker were purchased from TaKaRa Company; 2×EasyTaqPCRSuperMix 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 produced analytically pure.

[0040] 1.3 Kit

[0041] Total RNA extraction kits, PCR purification and gel extraction kits were purchased from QiaGen; 1stStrand cDNA...

Embodiment 2

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

[0141] Total RNA was extracted from mycelia, primordia and fruiting bodies of three different growth stages of Chizhi, reverse-transcribed using a reverse transcription kit (PROMEGA), and protein phosphatase 2A (PP2A) gene was used as an internal reference gene for fluorescent quantitative PCR analysis. The direction primer is: 5'-CTCGCACGCTACAACAAAG-3', the reverse primer is: 5'-AGTCGGAAGTGGGTGGG-3', the results show that the expression of the gene in mycelia is significantly higher than that in the primordium and fruiting body stage ( figure 1 ).

Embodiment 3

[0142] The prokaryotic expression of embodiment 3GL22395 gene cDNA sequence

[0143] 1. According to the full-length cDNA sequence of Chizhi terpenoid synthase, design primers for PCR amplification of the complete open reading frame, and add restriction enzyme sites BamHI and XholI 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, the reading frame of the red sesame terpene synthase gene is guaranteed to be completely correct, and the enzyme digestion reaction is carried out with BamHI and XholⅠ endonucleases, and the target fragment 1260bp is recovered; Escherichia coli expression vector pET28a is used BamHI and XholI endonucleases were digested to recover the target fragment 5kb. The target fragment of the chizhi terpene synthase gene that had been digested was clo...

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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 in particular relates to the cDNA sequence of a gene encoding chizhi terpene synthase GL22395 and its application. Background technique [0002] Ganoderma lucidum is a general term for fungi of the genus Ganoderma, Ganoderma lucidum, and G. sinense of the class Basidiomycetes, Polyproraceae. As a traditional medicinal fungus in my country, Ganoderma lucidum has been used for 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 "Shen Nong's Herbal Classic" more than 2,000 years ago. At present, Ganoderma lucidum has been included in "American Herbal Pharmacopoeia and Therapeutic Compendium" (American Herbal Pharmacopoeia and Therapeutic Compendium), and its medicinal value has been widely recognized by countries all over the world. . Ganoderma lucidum contains a variety of biologicall...

Claims

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Application Information

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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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