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Ganoderma lucidum terpene synthase GL-TS2 coding gene cDNA (complementary deoxyribonucleic acid) sequence and application of ganoderma lucidum terpene synthase GL-TS2 coding gene cDNA sequence

A GL-TS2, coding gene technology, applied in the field of genetic engineering, can solve the problems of difficult extraction and separation, unclear molecular mechanism, etc.

Active Publication Date: 2015-12-16
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 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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  • Ganoderma lucidum terpene synthase GL-TS2 coding gene cDNA (complementary deoxyribonucleic acid) sequence and application of ganoderma lucidum terpene synthase GL-TS2 coding gene cDNA sequence
  • Ganoderma lucidum terpene synthase GL-TS2 coding gene cDNA (complementary deoxyribonucleic acid) sequence and application of ganoderma lucidum terpene synthase GL-TS2 coding gene cDNA sequence
  • Ganoderma lucidum terpene synthase GL-TS2 coding gene cDNA (complementary deoxyribonucleic acid) sequence and application of ganoderma lucidum terpene synthase GL-TS2 coding gene cDNA sequence

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Embodiment 1

[0034] Example 1 Acquisition of the cDNA sequence of the gene encoding Chizhi terpene synthase GL-TS2

[0035]1. According to the measured Ganoderma lucidum genome (GenBank number: AGAX00000000.1) data, through operations such as splicing and annotation, obtain the cDNA sequence of the candidate terpene synthase gene.

[0036] 2. Take the vigorously growing mycelium of Ganoderma lucidum and use QiaGen The Mini kit was used to extract the RNA, and the reverse transcription kit (PROMEGA) was used to perform reverse transcription. The obtained cDNA was used as a template, and the primer design software LasergenePrimerSelect was used to analyze the cDNA sequence of the candidate gene. Before and after its open reading frame (ORF) Find the most suitable primer pair in the range of 100bp, and finally determine the primer sequence as:

[0037] Forward primer: 5′-GCCTCCCTCACCCCACTCCAT-3′

[0038] Reverse primer: 5′-GCGAGGACCACCTAAACCC-3′

[0039] Primers were synthesized by Beijin...

Embodiment 2

[0041] Example 2 Bioinformatics Analysis and Tissue Expression Analysis of GL-TS2 Gene cDNA Sequence

[0042] 1. The length of the open reading frame (ORF) of the red sesame terpene synthase GL-TS2 of the present invention is 1041 bp (Seq ID No. 2), encoding 346 amino acids (Seq ID No. 1). The GL-TS2 full-length open reading frame was searched for homology in the NCBI database using the BLAST program, and the BLASP comparison analysis of the gene at the amino acid level showed that the amino acid sequence of the protein encoded by the Chizhi GL-TS2 gene was similar to that of other species. The homology is high, and the similarity with Dichomitus squalens LYAD-421SS1 (Pyriophora filthii) which has been functionally verified is the highest, reaching 92%.

[0043] 2. Extract the total RNA of mycelia, primordia and fruiting bodies in three different growth stages of Chizhi, use the reverse transcription kit (PROMEGA) to perform reverse transcription, and use the protein phosphata...

Embodiment 3

[0044] Prokaryotic expression and functional analysis of embodiment 3GL-TS2 gene cDNA sequence

[0045] 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 NdeI and HindIII to the forward and reverse primers, respectively. The designed primers are:

[0046] Forward primer: 5′-CGCCATATGATGCCTGCA-3′

[0047] Reverse primer: 5′-CCCAAGCTTCTAGGCATG-3′

[0048] 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 NdeI and HindIII endonucleases, and the target fragment 1041bp is recovered; Escherichia coli expression vector pET28a is used NdeI and HindIII endonucleases were digested to recover the target fragment 5kb. The target fragment of the chizhi terpene synthase gene that had...

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Abstract

The invention relates to a ganoderma lucidum terpene synthase GL-TS2 coding gene cDNA (complementary deoxyribonucleic acid) sequence and application of the ganoderma lucidum terpene synthase GL-TS2 coding gene cDNA sequence. The ganoderma lucidum terpene synthase GL-TS3 coding gene cDNA sequence is shown as Seq ID No.2, and 346 amino acids are coded. Escherichia coli is converted through ganoderma lucidum terpene synthase gene GL-TS2; endogenous FPP (farnesyl pyrophosphate) is used as a substrate, and the goal of heterologous synthesis of terpenoid in the escherichia coli is achieved.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular, it relates to a cDNA sequence of a gene encoding chizhi terpene synthase GL-TS2 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 biologically a...

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李滢
Owner INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI
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