Cloning and characterization of antioxidation related genes of Antrodia cinnamomea
Date Issued
2007
Date
2007
Author(s)
Liu, Yu-Chen
DOI
zh-TW
Abstract
Antrodia cinnamomea (Neu-Chang-Tsu), an endemic resupinate basidiomycetes of Taiwan, inhabitated on the inner cavity of the endemic broad leaved tree, Cinnamomum camphoratum. The claimed potent medicinal activities, including of antioxidation, by A. cinnamomea were well recognized. Attempt to elucidate the antioxidation mechanism molecularly, experiments were initiated by blasting of the previously annotated A. cinnamomea cDNA library. Of the five ESTs, respectively encoding Cu-Zn superoxide dismutase, glutathione reductase, glutathione peroxidase, glutathione transferase, and cytochrome c peroxidase were accessed and cloned by the derived primers. Full-length cDNA of the genes were resolved by RACE (rapid amplification of cDNA ends). Moreover, the gDNA sequences corresponding to these five genes were gained by primer walking from the Fosmid clones shown positive hybridization signals against the DIG-labeled probes. The UTR, introns, active sites, conserved domains, and biochemical properties, etc. were defined by relevant bioinformatics websites. The resolved full-length of Cu-Zn SOD and cytochrome c peroxidase , the former consisting of 940 bp, with 561 bp ORF, 3 introns; while the later composed of 1456 bp, with 1116 bp ORF, and 7 introns.While the cDNA of glutathione peroxidase were 794 bp, 477 bp ORF, and 4 introns; glutathione transferase cDNA 727 bp, ORF 453 bp, and 6 introns; glutathione reductase cDNA 794 bp, ORF 453 bp, no intron. Compared to the EST or gDNA, presumably 20, 129, or 500 bp nucleotides of the three genes, were unallocated. Additionally, Cu-Zn SOD gene has been constructed in the E. coli expression vector pQE 31, and glutathione transferase gene in Saccharomyces cerevisiae complementation vector p426ADH, both neither expressed nor complemented properly, and need work further. With respect in expression of these five genes in mycelium or fruiting body of A. cinnamomea, virtual Northern(VN) or Q-PCR were performed. In general, the activities of these genes in mycelium were higher than those in fruiting body, by VN with the order SOD＞GP＞CYT＞GTR＞GR; while by Q-PCR, SOD＞GTR＞CYT＞GR＞GP.
Subjects
樟芝
抗氧化
超氧歧化酶
穀胱甘肽
相關酵素
Antrodia cinnamomea
antioxidation
superoxide dismutase
glutathione related enzyme
Type
other
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