Dissertation > Excellent graduate degree dissertation topics show

Molecular Cloning, Characterization, and Differential Expression of a Lanosterol Synthase Gene from the Fungus Ganoderma Lucidum

Author: ShangChangHua
Tutor: ZhaoMingWen
School: Nanjing Agricultural College
Course: Microbiology
Keywords: Ganoderma Triterpenoid Lanosterol synthase gene Functional verification Promoter Expression profiling
CLC: S567.31
Type: Master's thesis
Year: 2009
Downloads: 48
Quote: 0
Read: Download Dissertation

Abstract

Ganoderma (Ganoderma lucidum) in China has a long history of use, can be used for conditioning and treatment of many diseases. One of the main medicinal components of Ganoderma triterpenoids with anti-tumor effect, inhibiting histamine release of the anti-HIV and HIV protease inhibition of cholesterol synthesis and absorption. Therefore, the content of triterpenoids has become an important indicator to assess the medicinal value of Ganoderma lucidum. Triterpenoid as secondary metabolites by the mevalonate (MVA) pathway, after France ester pyrophosphate, squalene, squalane -2,3 - oxide and lanosterol alcohols such intermediates, and finally through a series of oxidation Restore as well as the formation of the cyclization reaction. Lanosterol synthase (LS) in the mevalonate (MVA) pathway of the second branching point, is a key enzyme in the synthesis process of the terpenoids, it can catalyze the 2,3 - oxidosqualene cyclize wool sterol. LS content, and activity level of production will affect the triterpenoids. Has been published in other species the the LS protein sequences conservative District, to design a pair of degenerate primers Ganoderma genomic DNA as a template for PCR amplification after degenerate primers, a 488bp Ganoderma LS gene-specific DNA fragments are obtained. Using the obtained gene-specific fragment, designed three specific primers, SEFA PCR method to obtain the 5 'end of the LS gene was amplified genomic DNA sequence. Ganoderma primordia Materials cDNA using this cDNA as a template, the 3'-RACE, the LS gene 3 'end of the cDNA sequence. PCR amplification of the sequence specific primers were designed to amplify, LS full-length cDNA sequence. The sequence length of 2181 bp, encoding 726 amino acids. Based on the obtained sequence information for stitching to obtain a full-length genome of the LS (genomic DNA) sequence. The sequence length of 2924 bp, LS gene is composed of 7 exons and 6 introns. The amino acid sequence was found Blast comparison, of Ganoderma LS basidiomycetes Cryptococcus neoformans Cryptococcus neoformans shows the highest similarity (53%), which is consistent with the classical taxonomy of Ganoderma lucidum. Ganoderma lucidum LS cDNA sequence inserted into the yeast expression vector pYES2 in into yeast mutants OSC △ deletion LS activity depends ergosterol (ergosterol) in order to survive. Results show that the transformants at 28 ℃, was not added to the medium of ergosterol (ergosterol) on growth, while the defect type haploid can not grow, confirmed that the gene obtained by the LS function. A pair of primers designed according to the sequence of the genome of the Ganoderma LS Ganoderma gene group DNA (The genomic DNA) as a template, amplification has been LS gene promoter sequence, the length of this sequence to 767bp, with PlantCARE on LS start promoter sequence to predict, results displayed LS gene promoter TATA-box and CAAT-box, as well as a number of cis-acting elements that respond to light, hormones ABA and environmental pressure factor. LS transcripts in different developmental stages of Ganoderma differences detected by real-time PCR method. The results show that in the Ganoderma lucidum primordium period, LS transcription of this quantity to be significantly higher than the number of transcripts in the mycelia. Such laws Hirotani et al reported the triterpenoid content variation. Signal substance added to the medium jasmonic acid methyl ester (methyl jasmonate, MeJA) can significantly induce Ganoderma Triterpene Synthesis. Therefore, MeJA treatment mycelium with different concentrations after which LS gene transcripts were detected. LS gene transcripts showing The continuity increases, the optimum induction concentration is 200 μm 100-200μM MeJA role. Meanwhile, the promoter activity analysis of the experiments show that there is at least a potential of methyl jasmonate responsive element in the promoter of the gene in the Ganoderma LS, the above results show that Ganoderma ls likely MeJA responsive element involved by MeJA mediated terpene biosynthesis.

Related Dissertations

CLC: > Agricultural Sciences > Crop > Economic crops > Medicinal crops > Fungus > Ganoderma lucidum
© 2012 www.DissertationTopic.Net Mobile