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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 許瑞祥(Ruey-Shyang Hseu) | |
| dc.contributor.author | Jiang-Fang Syu | en |
| dc.contributor.author | 許境芳 | zh_TW |
| dc.date.accessioned | 2021-06-14T16:41:41Z | - |
| dc.date.available | 2013-08-04 | |
| dc.date.copyright | 2008-08-04 | |
| dc.date.issued | 2008 | |
| dc.date.submitted | 2008-08-01 | |
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Lebenson Wiss Technol 31:664-667. 44. 陳志昇. 1999. 冬蟲夏草 Cordyceps sinsisis (Berk.) Sacc.分子生物鑑定系統之研究. 國立台灣大學農業化學研究所碩士論文. 45. 黃心穎. 2003. 以聚合酶鏈反應法檢測大豆及玉米中基因改造成分之研究. 國立台灣大學農業化學研究所碩士論文. 46. 楊怡真. 2008. 仙人掌桿菌群之分子檢測方法及非溶血性腸毒素表現之研究. 國立台灣大學生命科學院微生物與生化學研究所博士論文，台北. 47. 應建淅，卯曉嵐，馬啟明，宗毓臣，文華安. 1987. 中國藥用真菌圖鑒. 科學出版社，北京. 48. 謝秀欣. 2006. 巴西洋菇性別基因選殖與交配型之探討. 國立台灣大學微生物與生化學研究所碩士論文，台北. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/40137 | - |
| dc.description.abstract | 冬蟲夏草Cordyceps sinensis (Berk.) Sacc.為我國滋肺補腎的珍貴藥材，來源稀少、採集困難又無法進行人工栽培，因此價值逐年高漲。由於成本高昂，市售的冬蟲夏草產品多以人工培養無性世代的冬蟲夏草菌絲體取代野生的子實體。然而冬蟲夏草無性世代菌種中國被毛孢 (Hirsutella sinensis) 菌絲體的生長緩慢，導致來源有限，故常有以其他真菌菌絲體摻雜或替代於冬蟲夏草產品中。因此建立真正冬蟲夏草菌體含量的檢測系統為確保品質的首要課題。
本研究中首先比對已發表蟲草屬菌種性別基因中，較保守的alpha box與HMG box序列，設計簡併性引子 (degenerate primer) 擴增出冬蟲夏草的HMG box基因，由不同冬蟲夏草菌株的HMG box基因序列分析結果，設計僅可專一性擴增出冬蟲夏草HMG box基因的引子對 (HMGsp F/R)，經確定引子對的物種專一性、靈敏度以及結果再現性後，進行冬蟲夏草菌體即時定量系統的建立。在生活史推測部份，由於在不同冬蟲夏草菌株及子實體標本中只擴增出一種HMG box的性別基因，所以尚無法推測冬蟲夏草為雌雄同體型或雌雄異體型。但是由此HMG box序列所設計的專一性引子對(HMGsp F/R)針對冬蟲夏草具有極高的專一性，可作為核糖體基因外另一種鑑定冬蟲夏草的分生指標。以此專一性引子對 (HMGsp F/R)所建立的即時定量系統，可得HMG box基因拷貝數，再將拷貝數推算回原始總核酸量，最後帶入總核酸量與菌絲乾重的回歸方程式，即可推算出分析樣本中含有多少真正的冬蟲夏草。 | zh_TW |
| dc.description.abstract | Cordyceps sinensis (Berk.) Sacc is a very expensive medicinal fungus of Traditional Chinese Medicine, because it is hard to get and couldn’t process artificial propagation. If we would like to process artificial propagation, the first thing we have to know is life cycle of C. sinensis. So far, C. sinensis life cycle still unclear. This study would like to figure out whether C. sinensis belong to homothallism or heterothallism by mating gene. Due to C. sinensis is very expensive, Hirsutella sinensis mycelium was usually used to replace C. sinensis to make C. sinensis product. Since H. sinensis mycelium growth slowly leading cost higher, so products often mix another species mycelium or additive. It is very important to build quantitative system to detect the quality of C. sinensis products.
In the study, first is to design degenerate primer to clone alpha box and HMG box, conserve sequence in mating gene MAT1-1 and MAT1-2, and I obtained HMG box. Following, designed HMG box specific primer (HMGsp F/R). After chick the specific, sensitive and repeatability of this primer, and to build quantitative system by real-time PCR. In the part of presume C. sinensis life cycle, since I just obtained one mating gene in C. sinensis, so I couldn’t identify whether C. sinensis belong to homothallism or heterothallism, but due to the specific HMG box primer it could offer another molecular systematics of C. sinensis. Finally, using HMGsp F/R to build quantitative system could detect the quantity of H. sinensis mycelium successfully. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-14T16:41:41Z (GMT). No. of bitstreams: 1 ntu-97-R95b47407-1.pdf: 1537923 bytes, checksum: 80702f4631500e05300fda0c4d498a6a (MD5) Previous issue date: 2008 | en |
| dc.description.tableofcontents | 目錄 I
表目錄 IV 圖目錄 V 第一章 緒言 1 1 - 1蟲草屬簡介 1 1-1-1 蟲草屬的研究簡史 1 1-1-2 蟲草屬的分類地位 1 1-1-3 蟲草屬的感染途徑與形成 1 1-1-4 蟲草藥用： 2 1 - 2冬蟲夏草簡介 3 1-2-1 冬蟲夏草 3 1-2-2 冬蟲夏草的生長環境 4 1-2-3 冬蟲夏草的形態特徵 4 1-2-4. 冬蟲夏草無性世代分子生物鑑定法 4 1-2-5 冬蟲夏草分子生物定量系統的重要性 5 1 - 3 子囊菌交配系統 6 1-3-1 子囊菌非親和性系統介紹 6 1-3-2. 交配型 7 1-3-3. 子囊菌性別基因 7 1 - 4 即時定量聚合酶鏈反應分析 12 1-4-1 即時定量聚合酶鏈反應 12 1-4-2 相對定量與絕對定量 12 1-4-3 即時聚合酶鏈反應原理 13 1-4-4. 即時聚合鏈反應常使用的偵測化合物 14 1-4-5 即時聚合酶鏈反應偵測極限 15 1-4-6 即時聚合鏈反應應用範圍 16 五. 研究動機 17 第二章 冬蟲夏草性別基因及生活史探討 19 2 - 1 前言 19 2 - 2 材料 19 2-2-1 實驗菌株 19 2-2-2. 實驗質體 19 2-2-3. 實驗引子 19 2 - 3 實驗儀器 20 2 - 4 實驗套組 20 2 - 5 實驗方法 20 2-5-1. 菌種培養 20 2-5-2. DNA製備與純化 20 2-5-3. 冬蟲夏草鑑定 21 2-5-4 引子設計 21 2-5-5 DNA定量 22 2-5-6引子專一性分析 22 2-5-7 聚合酶鏈反應 22 2-5-8基因序列比較分析 23 2 - 6 結果 23 2-6-1 菌種確認 23 2-6-2 性別基因選殖 24 2-6-3 性別基因應用於冬蟲夏草分子生物鑑定 25 2 - 7 討論 25 三. 冬蟲夏草定量 27 3 - 1 前言 27 3 - 2 材料 27 3 - 3 實驗儀器 27 3 - 4 實驗套組 27 3 - 5 實驗方法 28 3-5-1. 菌種培養 28 3-5-2. DNA製備與純化 28 3-5-3 DNA定量 28 2-5-4 引子設計 28 3-5-5引子專一性分析 29 3-5-6以PCR測定引子對QF2/QR3偵測極限 29 3-5-7 Real-time PCR 29 3-5-8 標準參考物質之建構 29 3-5-9 菌體乾重對DNA量回歸曲線建立 30 3-5-10 即時聚合酶鏈反應準確性測試 30 3-6 結果 30 3-6-1 即時定量聚合酶鏈反應檢測系統的建立 31 3-6-2 標準曲線與PCR放大效率之計算 31 2-6-3 PCR與real-time PCR偵測極限比較 32 2-6-4 冬蟲夏草基因體大小推算結果 32 2-6-5 菌體乾重對DNA量回歸曲線建立 33 3-6-6 未知樣品冬蟲夏草含量推算 33 3-6-7 核酸總量與乾重實際值與即時聚合酶反應偵測值比較 34 3-6-8 干擾物是否會影響即時定量系統偵測結果 34 3 - 7 討論 35 第三章 結論 37 圖表 38 參考文獻 84 | |
| dc.language.iso | zh-TW | |
| dc.subject | 即時聚合酶 | zh_TW |
| dc.subject | 冬蟲夏草 | zh_TW |
| dc.subject | 中國被毛孢 | zh_TW |
| dc.subject | 性別基因 | zh_TW |
| dc.subject | 鏈反應 | zh_TW |
| dc.subject | Cordyceps sinensis | en |
| dc.subject | real-time PCR | en |
| dc.subject | mating gene | en |
| dc.subject | Hirsutella sinensis | en |
| dc.title | 以冬蟲夏草性別基因作為即時定量系統標的之研究 | zh_TW |
| dc.title | Using Cordyceps sinensis (Berk.) Sacc mating gene as target to build
real-time PCR detection system | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 96-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 黃慶燦(Ching-Tsan Huang),潘子明(Pan, Tzu-Ming) | |
| dc.subject.keyword | 冬蟲夏草,中國被毛孢,性別基因,即時聚合酶,鏈反應, | zh_TW |
| dc.subject.keyword | Cordyceps sinensis,Hirsutella sinensis,mating gene,real-time PCR, | en |
| dc.relation.page | 87 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2008-08-01 | |
| 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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