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研究生： 林文煜
Wen-Yu Lin
論文名稱： 利用核醣體內轉錄區間序列之種間特異性寡核苷酸探針快速檢測方法鑑定三種柴胡
Identification of three Bupleurum species through a rapid detection method using the sequence-specific oligonucleotide within ribosomal DNA internal transcribed spacer as probe
指導教授： 林彩雲 博士
Dr. Tsai-Yun Lin
口試委員:
學位類別： 碩士
Master
系所名稱： 生命科學暨醫學院 - 生物科技研究所
Biotechnology
論文出版年： 2004
畢業學年度： 92
語文別： 中文
論文頁數： 91
中文關鍵詞： 柴胡 、核醣體內轉錄區間序列 、種間特異性寡核苷酸探針 、中草藥基源鑑定 、親源關係 、轉漬膜
外文關鍵詞： Bupleurum, ribosomal DNA internal transcribed spacer, sequence-specific oligonucleotide probes, Authentication of Chinese medicinal herbs, phylogenesis, nylon membrane
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    • 囿於有限的外觀型態特徵及採收後多經加工與炮製等手續，實增添中草藥基源鑑定的困難性，因此本研究尋求並發展出一套利用分子生物技術於中草藥基源鑑定的快速檢測技術。柴胡為我國傳統醫藥中極為重要的肝膽保健及疾病治療之常用生藥，亦屬市售常誤用或混用中藥材之一。本論文含兩大部分，首先進行柴胡樣品親源關係的分析，使用的遺傳標記為細胞核核醣體DNA內轉錄區間序列，結果發現樣品間序列相似度很高、特異鹼基位置不多等特性。第二部分係以生物晶片原理為基礎，根據序列資料設計多組種間特異性寡核苷酸探針後，將探針固定於標準化轉漬膜上，鑑定高氏柴胡、三島柴胡及北柴胡。每個探針長度約15-20個鹼基，均含有一至三個種間特異鹼基。接著用Dig-11-dUTP分子標定以聚合脢連鎖反應擴增之核醣體DNA內轉錄區間片段，再與轉漬膜上種間特異性寡核苷酸探針進行雜交反應。偵測螢光訊號強、弱、有、無，可瞭解標的片段與探針黏合情形，進而鑑定待測樣品為何種柴胡，達到快速、精確、平價鑑定柴胡種原　的目的。本文實驗發現最佳雜交溫度約低於探針Tm 值10-13ºC間，在固定雜交溫度40ºC時，可一次獲得八組SSOP檢測結果 (即八處種間特異鹼基位置之鹼基資訊)，在六個柴胡乾品之四十八組SSOP訊號結果顯示，正確率達九成以上。日後若能以此檢測方法為基礎最佳化各項影響因素，將數種國內常見且外觀型態相似之中草藥乾品，或各種易混用、誤用中草藥SSOP同時整合固定於一標準化轉漬膜上，將可大大提高其實用性。

    Authentication of Chinese medicinal herbs gains more difficulties with the limitation of few unique morphological characters, and the process after harvesting. This study aims to develop an efficient method for rapid identification of Chinese medicinal herbs based on molecular biotechnology. Bupleuri Radix is a valuable and well known crude drug of Chinese medicine that is widely used in health care and remedy of liver disease. This herb is also one of the commonly misused crude drugs in Taiwan. The first part of this thesis is to analyze the genetic divergence among Bupleurum samples using the ribosomal DNA internal transcribed spacer (rDNA ITS) as marker. Our results showed that the ITS sequences of the three examined Bupleurum species share high identity with only few variations. In the second part, a rapid detection method was developed for identification of B. kaoi Liu Chao et Chuang, B. falcatum and B. chinense DC. based on oligonucleotide array. Oligomers of 15-20 bases within ITS containing one to three variation sites among different species were bound to nylon membrane. The ITS fragments were amplified with PCR using Dig-11-dUTP labeling and hybridized to a membrane holding the designed probes. The intensity of fluorescence signal was used to distinguish the binding strength between the amplified target sequence and the probe on membrane. Subsequently the suspected Bupleurum can be precisely identified through evaluation of the hybridization result in a short time and low-price. The appropriate hybridization temperature was measured below the Tm value of probe about 10-13ºC in our experiments. The signals of eight SSOP sets can be acquired after single reaction when we set hybridization temperature at 40ºC. The hybridization results of signals of forty-eight SSOPs with six dried Bupleurum samples displayed high accuracy (more than 90%). This method is reliable for authentication on condition that the factors of detection are optimized and sufficient SSOPs from the adulterate Chinese medicinal herbs are designed.

    目錄 中文摘要.....................................................................................................i 英文摘要...................................................................................................iii 謝誌………………………………………………………………………v 目錄...........................................................................................................vi 表目錄.....................................................................................................viii 圖目錄.......................................................................................................xi 名詞縮寫..................................................................................................xii 緒論............................................................................................................1 一、 前言……………………………………………………………..1 二、 柴胡分類與外形特徵…………………………………………..2 三、 以內轉錄區間核酸序列為遺傳標記…………………………..5 四、 寡核苷酸微陣列之轉漬膜檢測平台…………………………..7 材料與方法..............................................................................................11 一、 植物材料與維持………………………………………………11 二、 根尖細胞染色體觀察…………………………………………12 三、 基因組DNA抽取……………………………………………..13 四、 核糖體核酸的複製……………………………………………15 五、 核酸接合反應…………………………………………………16 六、 製備勝任細胞…………………………………………………16 七、 質體轉送至大腸桿菌…………………………………………17 八、 小量製備質體DNA…………………………………………...17 九、 雙股核酸自動序列定序………………………………………19 十、 親源關係樹的建立……………………………………………20 十一、 特異性寡核苷酸探針的製備…………………………..…21 十二、 探針固定於轉漬膜上………………………………….….22 十三、 PCR反應標定標的片段…………………………….….…23 十四、 雜交反應及訊號偵測……………………………………..23 結果..........................................................................................................26 一、 柴胡染色體數目觀察及基因組大小測量……………………26 二、 柴胡核醣體核酸的複製與構築………………………………26 三、 不同柴胡樣品核醣體核酸序列組成與特性比較……………26 四、 不同柴胡樣品ITS序列之遺傳歧異度分析…………………28 五、 三種柴胡序列與NCBI資料庫內其他柴胡屬物種序列分析.29 六、 以晉耆及北耆測試特異性寡核苷酸探針檢測方法…………30 七、 柴胡SSOP檢測方法初步試驗結果………………………….31 八、 建立檢測三種柴胡之標準化轉漬膜…………………………33 九、 柴胡乾品以標準化轉漬膜檢測結果…………………………34 討論..........................................................................................................37 參考資料..................................................................................................43 附錄一......................................................................................................91 表目錄 表一、三種柴胡型態特徵及特性比較....................................................47 表二、所設計的複製柴胡核醣體核酸之引子序列................................48 表三、標的片段 (PCR Dig-labeled target) 所使用之PCR標定反應液組成分及溫度程式…………………………………..................49 表四、八個柴胡樣品內轉錄區間核酸序列特性比較............................50 表五、於晉耆及北耆ITS1區域所設計SSOP序列.................................51 表六、複製晉耆及北耆基因組核酸ITS1片段所設計之引子序列.......51 表七、複製三種柴胡ITS1及ITS2片段之兩引子對序列......................52 表八、初步設計辨識三種柴胡之多組SSOP序列..................................53 表九、測試辨識三種柴胡之SSOP與標的片段雜交反應之最適溫度..54 表十、十二組SSOP序列組成情形....................................................…57 圖目錄 圖一、柴胡植株與癒傷組織。………………………………...……….58 圖二、柴胡乾品部分之外形和細部近拍。……….………….………..59 圖三、核醣體DNA結構。………………………………………...…….61圖四、北耆與晉耆內轉錄區間核酸序列排序情形。………...………...62 圖五、高氏柴胡（B. kaoi）、北柴胡（B. chinense）及三島柴胡（B. falcatum）內轉錄區間核酸序列排序情形。……………………64 圖六、高氏柴胡根尖細胞經染色後，於顯微鏡下觀察結果。…………66 圖七、以TCM1與TCM2引子進行PCR反應，複製柴胡核醣體核酸 所得產物之電泳圖。……………………………………………67 圖八、八個柴胡樣品內轉錄區間核酸序列排序情形。…………….….68 圖九、柴胡樣品間ITS序列相似度與相異度分析。…………………70 圖十、柴胡樣品及外群之ITS序列依Kimura-2-parameter鹼基取代模式計算所得之距離矩陣。………………………………………70 圖十一、柴胡樣品及外群之ITS序列利用neighber-joining（NJ）法所得到之樹形圖。………………………………………………71 圖十二、柴胡樣品及外群之ITS序列利用maximun parsimony（MP） 法所得到之樹形圖。…………………………………………72 圖十三、本文三種柴胡與自NCBI資料庫擷取之其他柴胡屬物種ITS 區域序列（連同三個外群序列）以neighber-joining法所繪得之樹形圖。………………………………………..…………..73 圖十四、晉耆及北耆基因組核酸ITS1片段以TCM3與Chi ITS1r引子進行PCR所得產物之電泳圖。……………..…………….74 圖十五、晉耆及北耆測試SSOP檢測方法所得結果。……..………….75 圖十六、以設計之兩引子對進行PCR反應複製三種柴胡基因組核酸ITS1及ITS2片段產物之電泳圖。……………..…………….76 圖十七、寡核苷酸探針ITS2_7 Bck以三種濃度固定於轉漬膜上與北柴胡標的片段雜交反應後所得訊號結果。…………………77 圖十八、初步設計辨識三種柴胡之SSOP與標的片段雜交反應結果。…………………………………………………….…..….78 圖十九、十二組SSOP分別與三種柴胡標的片段進行雜交反應之訊號結果。……………………………...………………………….79 圖二十、標準化轉漬膜上各組 SSOP探針設計情形。……….…….....81 圖二十一、設計之轉漬膜與三種柴胡標的片段進行雜交反應之預期訊號呈現態樣。…………………………..…………………..82 圖二十二、使用黃耆ITS區域標的片段與專為柴胡設計之轉漬膜進行雜交反應訊號結果。………………………………………83 圖二十三、以TCM3、TCM2引子進行PCR反應複製柴胡乾品核 醣體核酸產物之電泳圖。………………………..………..84 圖二十四、高氏柴胡【dried】標的片段與標準化轉漬膜雜交結果…..85 圖二十五、北柴胡【dried】標的片段與標準化轉漬膜雜交結果。……86 圖二十六、北柴胡【大陸湖北】標的片段與標準化轉漬膜雜交結果。..87 圖二十七、北柴胡【大陸深圳】標的片段與標準化轉漬膜雜交結果。…………………………………………………………88 圖二十八、北柴胡【台中-1】標的片段與標準化轉漬膜雜交結果。…...89 圖二十九、北柴胡【台中-2】標的片段與標準化轉漬膜雜交結果。…90

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