臺灣博碩士論文加值系統

蟲草屬真菌被作為傳統中藥已有很長的一段時間了，雖然蟲草屬真菌種類很多但僅有少數種類具有藥理功效，因此需要一個精確的方法來分析其藥理功效，目前已有人利用5.8S rDNA、28S rDNA、5S rDNA、18S rDNA、ITS、IGS區域的序列來做為分類上的依據，但這僅只能看見DNA的層級，並不能夠完整了解其基因的表現，因為藥理功效與代謝路徑及蛋白質的表現有關，本論文是第一次利用蛋白質體學技術來看不同蟲草屬真菌之蛋白質表現情形，論文中主要利用二維電泳技術來看八種不同蟲草屬分離菌株蛋白質之表現，這八株蟲草屬分離菌株分別是Cordyceps sinensis(冬蟲夏草BCRC C.S)、Cordyceps militaries (蛹蟲草，BCRC 32219)、Paecilomyces tenuipes(細腳擬青黴菌 IUM077、IUM067)和四株Beauveria amorpha (球孢白疆菌ROKI36、ROKI2229、IUM0922、BCRC35502)。每種菌株均以相同的培養環境包括溫度、pH、培養基轉速等，抽取蛋白質進行二維膠體電泳pH4-7 12.5％SDS-PAGE；接著以PDQuest軟體進行蛋白質圖譜的分析，結果顯示蛹蟲草和冬蟲夏草的圖譜幾乎完全不同，IUM67和IUM77有大約851個蛋白質點只有184個蛋白質點有相同的分子量大小(Mw)跟等電點(pI)、以ROKI36和ROKI2229、IUM0922、BCRC35502的圖譜比較分別有342、140、70個蛋白質點有相同的MW/pI；另外我們以統計學方式計算每對二維電泳圖譜的蛋白質點的相似度結果如下：BCRC32219和BCRC C.S相似度14％、IUM067和IUM077相似度12％、ROKI36和ROKI2229相似度24％、ROKI36和IUM0922相似度9％、ROKI36和BCRC 35502相似度4％，由於這些菌株有不同的宿主，所以儘管這些蟲草屬分離菌株核醣體DNA序列很相似，但因為宿主不同而使代謝路徑改變進而造成圖譜的差異，這也很可能是造成不同地區採集到的蟲草藥理活性不同的原因。

Cordyceps spp. have been used in the traditional Chinese medicine for a long time. Few medicinal Cordyceps spp. possess the curative effects. Therefore it is worth to identify the Cordyceps spp. with the pharmacological activities utilizing the accurate analytical method such as classification by 5.8S rDNA、28S rDNA、5S rDNA and 18S rDNA regions However, the rDNA identification can not distinguish the different pharmacological activities of Cordyceps spp..
In this study, we used two dimensional (2-D) gel electrophoreses to compare the proteomic profiles of 8 related isolates of the genus Cordyceps¾ Cordyceps sinensis BCRC C.S. 、Cordyceps militaries BCRC 32219、Paecilomyces tenuipes IUM077 、IUM067 、Beauveria amorpha ROKI2229、ROKI36、IUM0922 and BCRC35502. The 2-D gels were compared by PDQuest software. The comparative results demonstrated that the proteome map of C.S. was almost totally different from that of BCRC 32219. Among ~731 protein spots in IUM067 only 184 spots were found in IUM077 with the same pI and molecular weight. Compared to ROKI36, 342, 140 and 71 matched protein spots were found in ROKI2229, IUM0922 and BCRC35502 respectively. In order to estimate the similarities between the Cordyceps spp. isolates protein profile similarities were calculated between pairs of isolates using a matching co-efficient. The matching analyses results indicated that BCRC32219 with BCRC C.S similarity 14％, IUM067 with IUM077 similarity 12％, ROKI36 with ROKI2229 similarity 24％, ROKI36 with IUM922 similarity 9％ and ROKI36 with BCRC35502 similarity 4％, suggesting that the isolates of a species collected from the different host might have the different protein expressions even though they are supposed to have the same genome.

目次
第一章 前言 8
1.1背景介紹 8
1.2研究目的 8
第二章 文獻整理 10
2.1蟲草的介紹 10
2.1.1蟲草屬真菌的寄主 10
2.1.2蟲草的形成 10
2.1.3傳統藥用蟲草 11
2.2蟲草屬的療效 12
2.2.1呼吸系統 13
2.2.2心血管系統 13
2.2.3肝臟 14
2.2.4腎臟功能 14
2.2.5免疫系統 15
2.2.6抗氧化功能 16
2.2.7抗癌作用 17
2.2.9其他相關功能 17
2.3蟲草屬真菌的活性成分 18
2.3.1多醣類(polysaccharides) 18
2.3.2蟲草素(Cordycepin) 19
2.2.3固醇類(sterol) 19
2.2.4腺苷類(adenosine) 19
2.3 蟲草屬真菌的的多樣性 20
2.4蟲草屬真菌的分類 20
2.4.1傳統分類學 20
2.4.2科學分類方法 22
2.5 蛋白質體學 24
2.5.1 蛋白質體學的介紹 24
2.5.2 二維電泳技術 25
第三章 研究材料與方法 27
3.1 材料與藥品 27
3.1.1 人工培養液態發酵蟲草屬分離菌株之菌絲體 27
3.1.2 藥品 27
3.1.3 實驗器材 28
3.2 實驗方法 29
3.2.1蛋白質體學分析 29
第四章 結果 33
4.1 蟲草屬分離菌株二維電泳圖譜條件之建立 33
4.2 以二維電泳圖譜分析蟲草屬分離菌株液態發酵菌絲體之蛋白質圖譜 34
4.2.1 同屬不同種蟲草屬分離菌株菌絲體之蛋白質圖譜 34
4.2.2同屬同種蟲草屬菌絲體蛋白質圖譜 34
4.3蛋白質圖譜相似度分析 36
第五章 討論 38
第六章 參考文獻 42

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