臺灣博碩士論文加值系統

果實蠅科害蟲極具經濟重要性，尤其被列為檢疫害蟲種類，除在疫區造成之損害外，為防堵其擴散所耗費之人力物力更是驚人。在檢疫工作上，非疫區港口及田野之害蟲偵測是最基本的，而偵測工作中最重要的一環即是害蟲種類的鑑定。以分子方法鑑定昆蟲種類，具有操作簡便、流程標準、適用於生活史所有階段等優點，故在偵測工作上為一有力的工具。本實驗選取尚未入侵亞洲，但在世界其他地區造成嚴重危害且入侵潛力極高之地中海果實蠅，以及本地所產，最常見且危害最大並有擴散至其他地區可能之東方果實蠅、瓜實蠅及南瓜實蠅為材料，利用PCR-定序法取得其粒線體DNA序列資料，並據以設計種專一性引子對PCR的分子鑑定方法，目的在建立這四種重要果實蠅之粒線體DNA序列基礎資料，以及開發能適用於檢疫果實蠅偵測工作之分子鑑定方法。選為分子標記之粒線體DNA序列片段共有十段，扣除重疊部分總計9,260bp，約佔全粒線體基因組之3/5。所得序列經排列比較，能找到種專一性差異的區段；序列分析的結果，在十段粒線體DNA序列中，平均種內序列相似度 (98.01~99.33) 皆大於平均種間序列相似度(83.91%~94.31%)。以序列差異所建構之親緣關係樹亦與傳統分類相符。以所得序列資料所設計之專一性引子對PCR，經測試能用以鑑定此四種果實蠅，並適用於萃取所得DNA極少的情形。

Pest species in family Tephritidae are considered very important in economical aspect, especially those listed in quarantine pests. Since the damage caused in their endemic areas, the efforts of preventing these species from dispersing are great. The detection of quarantine pests at the harbors and in the field are essential in quarantine works, and the most important job of the detection is the identification of those pest species. Using molecular technique as a method of identifying insect species is easy for routine operation and its standardized procedure and application on all life stages make this method a powerful tool in the detection of quarantine pests. In this study, a major fruit fly species that has not invaded Asia but with high risk of invasion: Ceratitis capitata was chosen to be the material, along with three local tephritid pests: Bactrocera dorsalis, B. cucurbitae and B. tau, which are also the pests of highly economical importance. The aim of this study was to set up a basic molecular database which could be the foundation of designing species identification method and to actually design a simple identification method for the four species. By using PCR-sequencing, a total of 9,260bp mitochondrial DNA sequence data in ten molecular regions were resolved for all of these four species, that is about 3/5 of the total mitochondrial genome. After alignment of all the sequence data, species-specific sites were located. Analysis of the ten sets of sequence data showed that intraspecific sequence identity (98.01%~99.33%) are all higher than that in the interspecific sequences (83.91%~94.31%). And according to the results of phylogenetic analysis, the reconstructed relationships based on the sequence data basically matched that in the classical taxonomy. The species-specific molecular primers designed by sequence variations were also proven to be useful in distinguishing those four species, even under the conditions of the low extracted DNA amount.

中文摘要…………………………………………………………………i
英文摘要………………………………………………………………ii
目錄……………………………………………………………………iv
表次……………………………………………………………………vi
圖次……………………………………………………………………vii
壹、 前言…………………………………………………………1
貳、 往昔研究……………………………………………………3
一、 果實蠅的分類及經濟重要性………………………………3
二、 分子分類定義與常用技術…………………………………5
三、 粒線體DNA在分子分類上的特性……………………………6
四、 果實蠅分子分類研究現況…………………………………6
參、 材料與方法…………………………………………………10
一、 供試昆蟲……………………………………………………10
二、 粒線體DNA序列資料的取得與分析………………………10
（一） 基因組DNA之萃取…………………………………………10
（二） 保守性PCR…………………………………………………11
（三） 大腸桿菌質體選殖…………………………………………12
（四） 質體DNA之萃取……………………………………………13
（五） DNA定序與序列分析………………………………………13
三、 專一性引子對的設計與PCR反應…………………………13
肆、 結果…………………………………………………………14
一、供試昆蟲選擇及基因組DNA之萃取………………………………14
二、保守性PCR、大腸桿菌直體選殖及質體DNA萃取………………14
三、DNA序列分析………………………………………………………18
四、專一性引子對設計與PCR結果……………………………………39
伍、討論………………………………………………………………44
陸、引用文獻…………………………………………………………49
柒、附錄………………………………………………………………57
附錄一、四種果實蠅在十個分子標記片段完整序列………………57

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