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Molecular Cloning and Differential Expression of Isopentenyl-Diphosphate Delta Isomerase 1 Gene from the Fungus Ganoderma Lucidum

Author: YaoJian
Tutor: ZhaoMingWen
School: Nanjing Agricultural College
Course: Microbiology
Keywords: Ganoderma lucidum triterpenes isopentenyl-diphosphate delta-isomerase E. coli functional colour assay expression profiles
CLC: S567.31
Type: Master's thesis
Year: 2011
Downloads: 10
Quote: 0
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Abstract


Ganoderma lucidum (Fr.) Krast (Polyporaceae), one of the most famous traditional Chinese medicinal mushrooms, is a popular folk used to treat many diseases, such as hepatitis, hypertension, hypercholesterolemia and gastric cancer. Modern medical researches on this mushroom have demonstrated many interesting biological activities, including anti-tumor, inhibit the release of histamine, anti-HIV and inhibit cholesterol biosynthesis and so on. The quality of G. lucidum was mainy determined by the content of triterpenes.As a secondary metabolite, triterpenes are isoprenoids derived from isopenteny diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Synthesis of triterpenes occurs in the fungi, using IPP and DMAPP formed via mevalonate pathway. Isopentenyl-diphosphate isomerase (IDI) catalyzes a central reaction in the biosynthesis of isoprenoids. By converting IPP to its highly nucleophilic isomer DMAPP, it activates the basic isoprenoid unit for polymerization. Therefore, it is necessary for the synthesis of a wide variety of essential cellular metabolites.Based on the protein sequence alignment among the IDI1 from other species, degenerate primers were synthesized for PCR amplification of genomic DNA according to the conserved sequences. A 599 bp DNA fragment was obtained and sequenced. Three specific primers were designed on the basis of the specific 599 bp fragment of idil gene of G. lucidum. The 5’end and a 1202 bp promotor of Gl-idil gene were amplified using SEFA-PCR method. Total RNA was extracted from G. lucidum and reverse transcribed to cDNA. We used a 3’-RACE method to obtain the 3’end sequence of idil gene. Specific primers were synthesized based on the obtained sequence. The full length of cDNA was 759 bp encoding a 252 amino acid. The genomic sequence of G. lucidum idil was assembled and analyzed. The length of genomic DNA was 985 bp included 3 exons and 2 introns. The length of one intron is 59 bp, another is 167 bp. The functional colour assay indicated that G1IPI could accelerate the accumulation of p-carotene in Escherichia coli transformants. The cloning and functional analysis of GI-IDI1 will be useful in increasing understanding of the role of IPI in triterpenes biosynthesis at the molecular level in G. lucidum.Gl-IDI was predicted a protein with molecular mass of 28.70777kD and an isoelectric point of 5.36. In addition, by predicting the promoter sequences,17 CAAT box and 1 TATA box which are typical eukaryotic promoter elements were found. The potential regulatory elements such as fungal elicitor responsive element, MYB binding site and two methyl jasmonate (MeJA) responsive elements, Spl elements, LTR elements and ATC-motifs were also found in the promotor region.To examine different developmental stages and under the conditions of MeJA-induced the mRNA levels of the idil gene, we monitored the gene transcript levels by semi-quantitative RT-PCR. The results showed that the gene level of primordia was higher than the the mycelia in the mRNA level. Glidil expression was increased to amaximum level at 24 h after treatment with 254μM MeJA, declined thereafter.Besides, we expressed the G1IDI1 in E. coli systerm. And the obtained protein was purified by affinity chromatography using a Ni column. It will be helpful for teh future study.

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CLC: > Agricultural Sciences > Crop > Economic crops > Medicinal crops > Fungus > Ganoderma lucidum
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