臺灣博碩士論文加值系統

登革熱是目前全世界關注的疫情之一，而埃及斑蚊(Aedes aegypti)與白線斑蚊 (Aedes albopictus)則是登革熱病毒的主要兩大載體，他們對全世界人類的發病率及死亡率造成巨大影響。而台灣近幾年的登革熱疫情有逐漸往北移之趨勢，但是主要載體埃及斑蚊依然還是只分布於嘉義布袋以南區域，而白線斑蚊則分布於台灣全島平地及高度1500公尺以下山區，因此認為疫情的北移與白線斑蚊有極大關係。粒線體DNA序列可用於研究昆蟲種族之間族群分類之分析，因為粒線體DNA不會經過遺傳重組並且進化速率是細胞DNA的10倍，因此成為群體遺傳學研究中重要的標的。完整的粒線體序列包含了13個蛋白質編碼基因、22個tRNA和2個rRNA基因，以及1個A+T核酸含量極高的控制區( Control region )，此區擁有較快的突變速率，包含多個串聯的重複序列，負責粒線體基因組的複製，並含有轉錄起始的啟動子，常用來研究種系發生學。先前我們對白線斑蚊進行了散彈槍定序，並將結果與NCBI上白線斑蚊粒線體DNA序列做映射（reference mapping），結果除了序列變異之外，再參考序列中還發現了有一段約750 bp的序列完全無法映射上，而這段區域剛又位於粒線體的控制區。我們設計引子針對控制區和另外兩個常用來做系統發育分析的標的基因COI與ND5進行PCR定序。以COI與ND5兩個基因序列定序結果的分析可以成功鑑別出埃及斑蚊、白線斑蚊和淡色庫蚊三種蚊子的分類，而定序出的控制區序列可以明顯比較出白線斑蚊粒線體DNA此區的變異性極高，我的目標是完成白線斑蚊的粒線體全定序與註解分析，並且藉著高變異性的控制區的比較基因體學分析設計建立更詳細的族群分類。

Aedes albopictus and Aedes aegypti are major vectors of dengue virus, which causes a tremendous impact on human morbidity and mortality worldwide. Mitochondria DNA (mtDNA) sequences are useful in phylogenetic analysis of both inter and intraspecific studies among insects. With an evolution rate estimated 10 times faster than the nuclear genome, mtDNA variation became useful markers in population genetics studies. Previously we have performed shotgun sequencing of Ae. albopictus genome and the reads were mapped to reference sequence from NCBI ( GenBank AY072044 ). In addition to sequence variations, the control region of the reference mitochondria genome was not mapped by shotgun reads. The complete mitochondria genome was determined to contain 13 protein coding genes, 22 tRNA genes, and 2 rRNA genes cluster. The mitochondria DNA control region, also called “AT-rich region” due to its extremely high A+T nucleotide content (>83%), contained several tandem repeats. It is responsible for the replication of mitochondrial genome and contains the promoter for transcription initiation. Further sequencing of purified Ae. albopictus mtDNA revealed a 751bp sequence located in the control region that is missing in the reference. In addition to sequencing the mitochondrial genome control region, we also sequenced two important marker genes regions: the cytochrome c oxidase (COI) and NADH dehydrogenase subunit 5 (ND5) gene regions. Considerable variations in the control region was identified among the Ae. albopictus. Preliminary sequencing analyses and phylogenetic analyses based on the COI and ND5 sequences were performed among Ae. albopictus, Ae. aegypti and Culex quinquefasciatus. By exploring the variability of the mtDNA control region among Ae. albopictus population we aimed to establish the molecular basis to classify and monitor their population.

目錄
誌謝詞 i
中文摘要 ii
Abstract iii
表目錄 vi
圖目錄 vii
第一章前言 1
第一節登革熱的介紹 2
一、 登革病毒 2
二、 典型登革熱臨床表現和症狀 2
三、 登革出血熱及登革休克症候群 2
第二節斑蚊的介紹 3
一、 埃及斑蚊 3
二、 白線斑蚊 4
三、 埃及斑蚊與白線斑蚊的比較 5
第三節粒線體DNA的介紹 6
一、 群體遺傳學 7
二、 ND5與COI基因的介紹 7
三、 控制區(Control region)的介紹 8
第二章研究目的 9
第三章材料與方法 10
第一節 樣本收集與保存 10
第二節 溶液配置 10
第三節 培養基等藥品配置 11
第四節 粒線體抽取( 101Bio Mitochondrial Isolation Kit ) 11
第五節 DNA純化 12
第六節 PCR增幅目標DNA 13
第七節 電泳分析 17
第八節 PCR產物純化 17
第九節 PCR產物回收定序 17
第十節 序列比對、相關序列下載與序列分析 17
第四章結果與討論 19
第一節 Control region的Unmapped region 19
第二節 嘗試以genomic DNA進行mtDNA的控制區定序 19
第三節 各種蚊子的COI與ND5區段序列親源分析 20
第四節 利用mtDNA標記COI與ND5進行白線斑蚊的次群體組成的遺傳分析 21
第五節 實驗室白線斑蚊COI、ND5和Control region定序 23
第六節 白線斑蚊的Control region比較分析 24
第五章結論 26
第六章參考文獻 27

表目錄
表一埃及斑蚊與白線斑蚊生活習性比較 34
表二控制區Mapping 資料分析 34
表三引子的設計 35
表四台南蚊利用COI基因做蚊種分類 36
表五台南蚊利用ND5基因做蚊種分類 36
表六國衛院10隻台南蚊編號與COI, ND5 PCR定序結果，以及品種分類 37
表七全球白線斑蚊粒線體DNA COI 基因 38
表八全球白線斑蚊粒線體DNA ND5基因 39






圖目錄
圖一控制區裡unmapped region的發現 40
圖二參考序列( AY072044 )粒線體DNA片段 41
圖三控制區PCR結果電泳圖 42
圖四粒線體DNA裡COI基因PCR結果電泳圖 43
圖五粒線體DNA裡COI基因PCR結果電泳圖 43
圖六粒線體DNA裡COI基因PCR結果電泳圖 44
圖七粒線體DNA裡COI基因PCR結果電泳圖 44
圖八粒線體DNA裡ND5基因PCR結果電泳圖 45
圖九粒線體DNA裡ND5基因PCR結果電泳圖 45
圖十以COI gene進行本土環境中蚊子的親緣分析 46
圖十一以ND5 gene進行本土環境中蚊子的親緣分析 47
圖十二以COI序列進行白線斑蚊親緣樹的分析 48
圖十三以ND5序列進行白線斑蚊親緣樹的分析 49
圖十四粒線體DNA裡COI基因PCR結果電泳圖 50
圖十五粒線體DNA裡ND5基因PCR結果電泳圖 51
圖十六控制區PCR結果電泳圖 52
圖十七控制區PCR結果電泳圖 53
圖十八控制區PCR結果電泳圖 54
圖十九白線斑蚊粒線體complete sequence 組裝圖 55
圖二十參考序列( AY072044 )與實驗定序出的序列( ALBI )在粒線體DNA片段 56
圖二十一控制區的變異 57

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