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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 許瑞祥(Ruey-Shyang Hseu) | |
| dc.contributor.author | Chih-Sheng Chen | en |
| dc.contributor.author | 陳志昇 | zh_TW |
| dc.date.accessioned | 2021-06-15T04:25:16Z | - |
| dc.date.available | 2019-08-20 | |
| dc.date.copyright | 2009-09-02 | |
| dc.date.issued | 2009 | |
| dc.date.submitted | 2009-08-21 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/45527 | - |
| dc.description.abstract | 冬蟲夏草 (Cordyceps sinensis (Berk.) Sacc.) 自古為我國滋肺補腎的珍貴藥材，由於冬蟲夏草形態觀察鑑定不易，係多利用分子生物技術分析核醣體RNA基因(nuclear ribosomal RNA gene，簡稱nrRNA基因或nrDNA) 18S及內轉錄區ITS1 (internal transcribed spacers 1)、5.8S、ITS2序列作為分類鑑定指標。為解決冬蟲夏草相關nrDNA分類文獻中「複合種」(species complex)、「隱蔽種」(cryptic species) 觀點分歧的現況，本試驗設計多組引子組來擴增供試樣品nrDNA，發現GC、AT-1、AT-2、T等四型nrDNA，且確認每個冬蟲夏草樣品同時具有GC型及另一型nrDNA。本研究結果證明了「單一物種同時具有兩種nrDNA」的現象。此結果可以合理解決兩種觀點的分歧，亦是未來冬蟲夏草分類鑑定上須考量的重要環節，至於各型樣品是否有藥理功能上或地理分佈上的差異，則有待未來進一步研究討論。 | zh_TW |
| dc.description.abstract | Cordyceps sinensis (Berk.) Sacc. is a valued traditional Chinese medicinal fungus. Since it is difficult to identify C. sinensis based on morphological observation, sequences of the 18S and internal transcribed spacer (ITS) regions (ITS1, 5.8S and ITS2) of nuclear ribosomal DNA (nrDNA) have been used to differentiate C. sinensis specimens. In the present study, we designed several primer pairs to amplify the nrDNA of C. sinensis isolates. A novel discovery was that the C. sinensis isolates had four types of nrDNA: GC, AT-1, AT-2 and T. Each isolate contained the GC type and simultaneously contained a further one type. These results provide a novel concept, i.e. the ‘multiple nrDNA type concept’ of C. sinensis classification, which differs from the previous ‘species complex’ and ‘cryptic species’ concept. Accordingly, we propose that the classification of C. sinensis based on nrDNA sequences should take into consideration the composition of nrDNA types. However, the relationship between the medicinal effects and geographical populations with different nrDNA profiles needs further study. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T04:25:16Z (GMT). No. of bitstreams: 1 ntu-98-D88623804-1.pdf: 10684233 bytes, checksum: 40cf8f1ed97d298967b4ce50605728dc (MD5) Previous issue date: 2009 | en |
| dc.description.tableofcontents | 目 錄
圖目錄 ………………………………………………………………………......... V 表目錄 ………………………………………………………………………......... IX 壹、 緒論 ………………………………………………………………………. 1 一、 核醣體簡介 ……………………………………………………………….. 1 1. 核醣體的構造與組成 ………………………………………………..... 1 2. 核醣體的功能－蛋白質合成 …………………………………………. 5 3. 核醣體與蛋白質合成抑制物 …………………………………………. 9 二、 核醣體RNA簡介 ………………………………………………………… 11 1. 核醣體RNA的功能 …………………………………………………… 11 2. 核醣體RNA基因應用於物種分類特點 ……………………………… 15 三、 蟲草屬簡介 ……………………………………………………………….. 19 1. 蟲草屬的研究簡史 ……………………………………………………. 19 2. 蟲草屬的分類地位 ……………………………………………………. 19 3. 蟲草屬有性世代的形態特徵 …………………………………………. 22 4. 蟲草屬無性世代及形態特徵 …………………………………………. 23 5. 蟲草屬的寄主 …………………………………………………………. 28 6. 蟲草屬感染寄主的途徑 ………………………………….…………… 28 7. 蟲草屬相關分子生物分類研究 ………………………………………. 29 8. 蟲草屬的應用 …………………………………………………………. 30 四、 冬蟲夏草簡介 ..………………………………………………………….. 36 1. 冬蟲夏草的分類地位及形態特徵 ………………………………......... 39 2. 冬蟲夏草相關研究史 …………………………………………………. 44 3. 冬蟲夏草的藥理功能 ……………………………………………......... 45 4. 冬蟲夏草無性世代菌種的發現 ………………………………………. 47 5. 冬蟲夏草無性世代菌種的形態特徵 …………………………………. 49 6. 冬蟲夏草無性世代菌株的產品開發 …………………………………. 53 7. 冬蟲夏草菌絲體醱酵產品之活性成分及藥理功能 …………………. 54 8. 冬蟲夏草及其菌絲體醱酵產品的市場價值 …………………………. 55 9. 以分子生物學指標鑑定冬蟲夏草無性世代菌種的結果 ……………. 55 五、 研究動機 ………………………………………………………………..… 62 貳、 材料與方法 ………………………………………………………………. 63 一、 試驗標本及菌株 ……………………………………………………………. 63 二、 菌種培養 ……………………………………………………………………. 63 三、 試驗樣品處理 63 四、 菌絲細胞核數目觀察 ………………………………………………………. 64 五、 DNA萃取 …………………………………………………………………… 68 六、 聚合酶鍊鎖反應 …………………………………….……………................ 68 七、 PCR擴增產物選殖到 yT&A載體 ………………………………………… 72 八、 限制酶水解多型性圖譜 ………………………............................................. 72 九、 核酸定序 ……………………………………………………………………. 72 十、 nrDNA序列分析與譜系關係計算 ………………................................……. 73 參、 結果 ………………………………………………………………………. 84 一、 冬蟲夏草樣品nrDNA ITS1、5.8S、ITS2區間定序結果 ……………......... 84 二、 冬蟲夏草樣品nrDNA ITS1、5.8S、ITS2區間序列分析結果 ………......... 100 三、 冬蟲夏草nrDNA ITS1、5.8S、ITS2 區間序列與GenBank基因資料庫核酸序列比對結果 ………………………………………...……………... 104 四、 冬蟲夏草18S nrDNA序列片段與GenBank基因資料庫序列比對結果 ….............................................................................................................. 109 五、 冬蟲夏草不同型nrDNA ITS1、5.8S、ITS2區間序列之分析 ………..…... 111 六、 冬蟲夏草不同型18S nrDNA序列分析之結果 …………………………. 128 七、 冬蟲夏草不同型nrDNA之複製倍數比例 ………………………………. 131 八、 冬蟲夏草不同型nrDNA序列源自於單一菌種之說明 …………………. 133 肆、 討論 ………………………………………………………………………. 135 一、 單一物種具多型nrDNA在生物學上的意義 ……………………………. 135 二、 冬蟲夏草多型nrDNA的發現 …………….…………………………….... 136 三、 nrDNA序列GC含量差異所代表的意義 ………………………………... 138 四、 以18S nrDNA NS3/NS6區間鑑定冬蟲夏草的沿革 ……………………. 139 伍、 結論 ………………………………………………………………………. 140 陸、 未來研究工作 ……………………………………………………………. 141 柒、 參考文獻 …………………………………………………………………. 142 附錄1、冬蟲夏草不同型nrDNA序列片段 ……………………………………. 157 附錄2、本論文內容已發表於國際期刊之文章 ………………………………… 163 附錄3、本論文內容已發表於國內期刊之文章 ………………………………… 172 圖目錄 圖1. 細菌及真核細胞的核醣體 ………………………………………………... 3 圖2. 真核細胞轉譯起始步驟 …………………………………………………... 6 圖3. 細菌轉譯延長步驟 ………………………………………………………... 7 圖4. 細菌轉譯終止步驟 ………………………………………………………... 8 圖5. 大腸桿菌rRNA及部份核醣體蛋白質於核醣體之分佈位置圖 …………. 12 圖6. 脊椎動物rRNA轉錄過程 …………………………………………………. 13 圖7. 細菌rRNA轉錄過程 ………………………………………………………. 14 圖8. 大腸桿菌Escherichia coli 16S rRNA所推測的二級結構 ……………….. 17 圖9. 麵包酵母18S rRNA所推測的二級結構 …………………………………. 18 圖10. 蟲草屬子座的形態 ...…………………………………………………….. 24 圖11. 蟲草屬子囊殼的形態 ………………………………...…………………... 25 圖12. 蟲草屬子囊孢子的形態 …………………………………………………. 26 圖13. 蟲草屬的三個亞屬 ………………………………………………………. 27 圖14. 本試驗使用的冬蟲夏草標本 ……………………………………………. 32 圖15. 冬蟲夏草 ………………………………………………………………... 33 圖16. 蛹蟲草 ……………………………………………………………………. 34 圖17. 涼山蟲草 …………………………………………………………………. 34 圖18. 中國冬蟲夏草的分佈地區 ……………………………………………… 37 圖19. 世界上冬蟲夏草的分佈地區 ……………………………………………. 38 圖20. 冬蟲夏草模式標本的形態特徵 ……………...………………………….. 41 圖21. 冬蟲夏草子座的形態特徵 ……………...……………………………….. 43 圖22. 中國被毛孢的形態特徵 ……………...………………………………….. 52 圖23. (a) 複合種與 (b) 隱蔽種示意圖 ……………….……………………….. 60 圖24. 引子位置及其延伸方向示意圖 …………………………………………. 70 圖25. 冬蟲夏草及蟲草屬樣品nrDNA ITS1、5.8S、ITS2區間序列比對結果 … 88 圖26. 以最高簡約分析法分析本試驗冬蟲夏草樣品之nrDNA ITS1、5.8S、ITS2區間序列所得譜系關係圖 ……………….………………………... 102 圖27. 本試驗冬蟲夏草樣品Cs7528A2、RS3、W1023t與中國被毛孢模式標本HMAS 55469 nrDNA ITS1、5.8S、ITS2區間序列比對結果 ……........ 103 圖28. 以鄰近連接分析法分析冬蟲夏草及其相關真菌nrDNA序列ITS1、5.8S、ITS2區間所得譜系關係圖 ……………….………………………. 108 圖29. 本試驗設計的引子及其延伸方向示意圖 …………….………………… 112 圖30. 冬蟲夏草樣品不同型nrDNA序列ITS1、5.8S、ITS2區間序列示意圖 … 116 圖31. 以鄰近連接分析法分析冬蟲夏草及其相關真菌nrDNA序列ITS1、5.8S、ITS2區間所得譜系關係圖 ..…………………….……………… 118 圖32. 以最高簡約分析法分析冬蟲夏草及其相關真菌nrDNA ITS1、5.8S、ITS2區間序列所得譜系關係圖 ……………….…………….................. 123 圖33. 以最大似然性分析法分析冬蟲夏草及其相關真菌nrDNA ITS1、5.8S、ITS2區間序列所得譜系關係圖 ...………….................………………… 125 圖34. 以貝氏導出式分析法分析冬蟲夏草及其相關真菌nrDNA ITS1、5.8S、ITS2區間序列所得譜系關係圖 …………………...………….………… 127 圖35. 冬蟲夏草樣品不同型nrDNA 18S區間示意圖 ……….………………… 129 圖36. 以引子組 (A) GC18NS7/GCITS4 (GC型)、(B) AT1218NS7/AT12ITS4 (AT-2型) 擴增冬蟲夏草標本Cs7528A2之nrDNA所得電泳圖 …........ 132 圖37. 冬蟲夏草菌種RS3之 (A) 單核菌絲體、(B) 雙核菌絲體 ……...……… 134 表目錄 表1. 核醣體的分類與組成 ……………………………………………………... 4 表2. 蛋白質生合成抑制物 ……………………………………………………... 9 表3. 有關蟲草屬的典籍資料 ……………………………………….………….. 20 表4. 蟲草屬菌種的藥用價值 ………………………………………...………… 35 表5. 有關冬蟲夏草藥理的典籍資料 ………………………...………………… 46 表6. 本試驗所用蟲草屬樣品 ………………………………...………………… 65 表7. 本試驗所使用之引子 …………………………………...………………… 71 表8. 本試驗自GenBank基因資料庫引用的蟲草屬菌種序列 ……………… 74 表9. 冬蟲夏草樣品在基因資料庫的登錄編號 …………...…………………… 85 表10. 冬蟲夏草18S nrDNA NS5/NS6區間序列相同度 ……………………… 110 表11. 本試驗所設計的引子 ……………………………….…………………… 113 表12. 本試驗用於擴增不同型nrDNA序列的冬蟲夏草樣品 ………………… 115 表13. 冬蟲夏草不同型nrDNA ITS1、5.8S、ITS2區間的序列相同度 ………… 121 表14 蟲夏草不同型18S nrDNA序列片段相同度 …………….……………… 130 | |
| dc.language.iso | zh-TW | |
| dc.subject | 冬蟲夏草 | zh_TW |
| dc.subject | 複合種 | zh_TW |
| dc.subject | 核醣體RNA基因 | zh_TW |
| dc.subject | 隱蔽種 | zh_TW |
| dc.subject | nrDNA | en |
| dc.subject | Cordyceps sinensis | en |
| dc.subject | Cryptic species | en |
| dc.subject | Species complex | en |
| dc.title | 藥用真菌冬蟲夏草多型18S、ITS區域核醣體RNA基因序列之研究 | zh_TW |
| dc.title | Study on Multiple Types of 18S and ITS Region nrDNA in the Medicinal Fungus Cordyceps sinensis | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 97-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 簡秋源,黃慶璨(Ching-Tsan Huang),楊盛行,蘇慶華 | |
| dc.subject.keyword | 冬蟲夏草,核醣體RNA基因,複合種,隱蔽種, | zh_TW |
| dc.subject.keyword | Cordyceps sinensis,nrDNA,Species complex,Cryptic species, | en |
| dc.relation.page | 178 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2009-08-21 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 微生物與生化學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
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