曾顯雄臺灣大學：植物病理與微生物學研究所劉宇真Liu, Yu-ChenYu-ChenLiu2007-11-272018-06-292007-11-272018-06-292007http://ntur.lib.ntu.edu.tw//handle/246246/58020牛樟芝(Antrodia cinnamomea)為台灣特有之平伏型擔子菌，生長於台灣特有種牛樟樹之樹心空胴內壁，民間盛傳此菇具多重生理活性，抗氧化即為其中之一。為藉由分子層次闡明其抗氧化機制，遂進行篩檢應用真菌研究室所建構、註解之牛樟芝cDNA基因資料庫，由尋獲之五個和抗氧化相關之基因片段EST設計引子對去選殖Cu-Zn superoxide dismutase，Glutathione reductase，Glutathione peroxidase，Glutathione transferase，以及Cytochrome c peroxidase等基因。利用RACE (rapid amplification of cDNA ends)求取此等基因之cDNA全長，以及將對此等基因探針呈雜合反應之Fosmid clones進行primer walking定序，以求取其gDNA序列。已選殖出Cu-Zn superoxide dismutase以及Cytochrome c peroxidase基因之全長度cDNA，前者長度為940 bp，其ORF為591 bp，具3個intron；後者長度為1456 bp，其ORF為1116 bp，具7個intron。而所選殖之glutathione peroxidase其cDNA長為794 bp，ORF為477 bp，具4個intron；glutathione transferase其cDNA長為727 bp，ORF為456 bp，具6個intron；glutathione reductase其cDNA長為794 bp，ORF為453 bp。此三個基因和牛樟芝之EST及gDNA相比，分別可能有20，129，或500 bp之鹼基對尚未被求出。此等基因之生化特性亦經由相關之生物資訊網站加以分析。Cu-Zn SOD已建構於E. coli表現載體pQE 31，但表現不佳；Glutathione transferase也已將其建構於Saccharomyces cerevisiae之互補表現載體p426ADH，以相對之酵母菌S. c.突變株進行互補，亦尚未獲得成功之轉型株，未來將再繼續探討。此外，也利用Virtual Northern(VN)及Q-PCR比較此五個抗氧化基因於牛樟芝菌絲體或子實體之相對表現。一般而言，結果顯示菌絲之表現高於子實體；而分別就不同基因，以VN之結果分析，相對表現量之大小順序為SOD＞GP＞CYT＞GTR＞GR；而就Q-PCR結果分析，則為SOD＞GTR＞CYT＞GR＞GP。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.中文摘要------------------------------------------------------------------------------------------- 1 Abstract -------------------------------------------------------------------------------------------- 2 前言------------------------------------------------------------------------------------------------- 4 壹、 前人研究------------------------------------------------------------------------------------- 6 牛樟芝簡介--------------------------------------------------------------------------------------- 6 （一） 分類地位---------------------------------------------------------------------------- 6 （二） 型態特性----------------------------------------------------------------------------- 7 （三） 一般成分分析----------------------------------------------------------------------- 8 （四） 生物功能活性研究--------------------------------------------------------------- 10 （五） 牛樟芝之毒理分析--------------------------------------------------------------- 15 自由基的生成----------------------------------------------------------------------------------- 16 生物抗氧化相關機制-------------------------------------------------------------------------- 17 超氧歧化酶(superoxide dismutase, SOD)-------------------------------------------------- 17 （一） 銅鋅型超氧歧化酶(Cu-Zn SOD)----------------------------------------------- 18 （二） 錳型超氧歧化酶(Mn-SOD)----------------------------------------------------- 19 （三） 鐵型超氧歧化酶(Fe-SOD)------------------------------------------------------ 19 麩胺酸生合成途徑(glutathione synthesis pathway)與相關生化反應----------------- 20 （一） 麩胺酸還原酶(glutathione reductase)------------------------------------------ 21 （二）麩胺酸過氧化酶(glutathione peroxidase)-------------------------------------- 22 （三）麩胺酸轉移酶(glutathione transferase)----------------------------------------- 24 細胞色素c過氧化酶(cytochrome c peroxidase)------------------------------------------- 26貳、材料與方法---------------------------------------------------------------------------------- 28 一、 菌株及其培養----------------------------------------------------------------------- 28 二、 RNA製備---------------------------------------------------------------------------- 28 三、 反轉錄合成cDNA----------------------------------------------------------------- 31 四、 同步定量PCR----------------------------------------------------------------------- 31 五、 虛擬北方氏雜合反應-------------------------------------------------------------- 33 六、 抗氧化相關基因之RACE（Rapid Amplification of cDNA Ends）---------- 40 七、 牛樟芝Fosmid library中抗氧化相關基因之clone篩選及定序------------ 41 八、 Fosmid primer walking ------------------------------------------------------------ 44 九、 利用大腸桿菌表現系統進行基因功能性分析-------------------------------- 45 十、 酵母菌突變株互補實驗進行基因功能性分析-------------------------------- 47 十一、 序列分析 ----------------------------------------------------------------------- 47 參、結果------------------------------------------------------------------------------------------- 49 （一）麩胺酸還原酶 (glutathione reductase) ---------------------------------------- 49 （二）麩胺酸過氧化酶 (glutathione peroxidase) ------------------------------------ 53 （三）麩胺酸轉移酶 (glutathione S-transferase) ------------------------------------ 56 （四）細胞色素c過氧化酶 (cytochrome c peroxidase) --------------------------- 59 （五）超氧歧化酶 (superoxide dismutase) ------------------------------------------- 62 肆、討論------------------------------------------------------------------------------------------- 67 伍、圖表------------------------------------------------------------------------------------------- 74 陸、參考文獻------------------------------------------------------------------------------------ 125 附錄一、實驗試劑配方----------------------------------------------------------------------- 138 附錄二、培養基成分-------------------------------------------------------------------------- 141 附錄三 -------------------------------------------------------------------------------------------142 附錄四 -------------------------------------------------------------------------------------------1484246518 bytesapplication/pdfen-US樟芝抗氧化超氧歧化酶穀胱甘肽相關酵素Antrodia cinnamomeaantioxidationsuperoxide dismutaseglutathione related enzymeotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/58020/1/ntu-96-R93633001-1.pdf