臺灣博碩士論文加值系統

沃巴赫氏菌Wolbachia pipients是一種α-變形菌綱立克次體科的絕對性細胞內共生菌，廣泛存在於昆蟲與節肢動物體內，此內共生菌會造成宿主多種的生殖異常現象，包括細胞質不親和性（cytoplasmic incompatibility）、子代雌性化（feminization）、殺雄性（male killing）以及孤雌生殖（parthenogenesis）。迄今，這些生殖異常現象之成因，仍未有詳盡的解答。Negri et al. (2009)的研究報告指出，Wolbachia可能藉由造成葉蟬Z. pullula的染色體甲基化，而使受感染的葉蟬的雄性後代會發育出雌性特徵。Wolbachia甲基轉移酶( methyl transferase)可能造成Wolbachia在宿主發生的生殖異常現象。因此，在本研究中，欲選殖綠豆象(Callosobruchus chinensis)及米象(Sitophilus oryzae Linnaeus)體內的Wolbachia甲基轉移酶，並分別命名為Cch_met以及Sli_met。利用RT-PCR檢測Wolbachia甲基轉移酶在成蟲體內的表現情形，結果顯示此基因於雌、雄體內都會表現，然而不同齡期的雌雄表現量是有所差異的。以pGEM- T easy vector進行Wolbachia甲基轉移酶蛋白質表現，並以西方墨點法檢測，證實Wolbachia甲基轉移酶可在大腸桿菌BL21(DE3)中表現，且Wolbachia甲基轉移酶也會造成大腸桿菌染色體發生甲基化改變的現象。

Wolbachia is one kind of obligated cellular symbiont belongs to alpha Proteobacteria, Rickettsiales and is widely found in insect and arthropod.This intracellular symbiont can cause many reproductive variation of its host,that including male killing, feminization , parthenogenesisand cytoplasmic incompatibility. So far, the causes of these phenomena were not cleard. Wolbachia may be caused methylation by the leafhopper genome., and determined the infected male offspring of the leaf hopper will development female characteristics. This study use the Callosobruchus chinensis and Sitophilus oryzae Linnaeus as model animals and cloned the full-length of Wolbachia DNA methyl transferase in Wolbachia, named Cch_Met and Sli_Met. Furthermore, it is seen the appearance of different mRNA expression of Wolbachia DNA methyltransferase between sexs by RT-PCR ,and the higher expression in female can be observed more than male. Protein expression assay DNA methyltransferase of the pGEM vector, and confirmed the DNA methyltransferase in E. coli BL21 (DE3) by Western blot. Then, methylation of the Wolbachia DNA methyltransferase in E. coli BL21 (DE3) chromosome by isoschizomers (DE3) , and showed methylation in E. coli chromosome.

致謝……………………………………………………………………Ⅰ
中文摘要………………………………………………………………Ⅱ
英文摘要………………………………………………………………Ⅲ
目錄……………………………………………………………………Ⅴ
圖目錄…………………………………………………………………Ⅵ
表目錄…………………………………………………………………Ⅷ
附錄……………………………………………………………………Ⅸ
縮寫檢索表 ……………...……………………………………………Ⅹ
前言………………………………………………………………………1
材料與方法……………………………………………………………12
結果……………………………………………………………………25
討論……………………………………………………………………32
結論……………………………………………………………………38
參考文獻……………………………………………………………… 39
 
圖 目 錄
圖一、wPip DNA methyl transferase wpa_0258基因組………………44 圖二、wpa_0258上游基因wpa_0259的胺基酸序列比對……………45
圖三、wpa_0257的胺基酸序列比對…………………………………46
圖四、豆象Wolbachia甲基轉移酶wpa_0258…………………………47
圖五、米象Wolbachia甲基轉移酶………………………………….…48
圖六、豆象Wolbachia甲基轉移酶之選殖……………………………49
圖七、選殖pGEMT之米象Wolbachia甲基轉移酶……………………50
圖八、核苷酸定序之豆象Wolbachia甲基轉移酶……………………52
圖九、米象Wolbachia甲基轉移酶之核苷酸定序……………………53
圖十、豆象、米象Wolbachia甲基轉移酶胺基酸序列比對……………55
圖十一、設計RT-PCR豆象甲基轉移酶專一性引子…………………56
圖十二、米象甲基轉移酶RT-PCR專一性引子之設計………………57
圖十三、豆象甲基轉移酶的mRNA 在不同時期間的表現量………58
圖十四、米象Wolbachia甲基轉移酶的mRNA表現量………………60
圖十五、豆象甲基轉移酶在雌性及雄性的mRNA表現量比例………62
圖十六、豆象甲基轉移酶在雌性及雄性的mRNA表現量比例………63
圖十七、豆象甲基轉移酶蛋白質表現專一性引子……………………64
圖十八、米象甲基轉移酶蛋白質表現專一性引子……………………65
圖十九、豆象甲基轉移酶重組蛋白之PCR產物………………………67
圖二十、米象甲基轉移酶重組蛋白之PCR產物………………………68
圖二十一、甲基轉移酶之PCR產物送入的pGEM-Teasy載體………69
圖二十二、DNA甲基轉移酶之蛋白質表現…………………………70
圖二十三、DNA甲基轉移酶之蛋白質表現…………………………72
圖二十四、Western blot分析DNA甲基轉移酶之蛋白質表現………73
圖二十五、豆象甲基轉移酶重組蛋白的活性分析……………………75
圖二十六、活性分析米象甲基轉移酶的重組蛋白……………………77
圖二十七、豆象、米象甲基轉移酶的重組蛋白活性分析……………79
圖二十八、未經IPTG誘導之甲基轉移酶重組蛋白活性分析…………80
圖二十九、建構pACYC-米象Wolbachia甲基轉移酶之載體………81
圖三十、選殖pACYC-米象Wolbachia甲基轉移酶…………………82
圖三十一、選殖pACYC-Met& pET-TFs之共表現載體………………83
圖三十二、pACYC-Met & pET-TFs活性分析…………………………85
圖三十三、pACYC-Met & pET-TFs活性分析…………………………87
圖三十四、Wolbachia甲基轉移酶親緣關係樹圖……………………88

表目錄
表一、Cch_met選殖的專一性引子……………………………………89
表二、Sli_met選殖的專一性引子………………………………………90
表三、Wolbachia甲基轉移酶基因選殖之PCR條件…………………91
表四、共表現載體的活性分析之PCR條件……………………………92

 
附錄
附錄一、Map of pGEM-T easy vector……………………………….…93
附錄二、Expresso™ T7 Cloning and Expression Syste…………94
附錄三、Map of pACYC vector……………………………………… 95

 
縮寫檢索表
錨蛋白重複序列Ankyrin repeat domain (Ank)
豆象甲基轉移酶 Callosobruchus chinensis (Cch_met)
細胞週期調控甲基轉移酶Cell cycle-regulated methyl transferase (Ccrm)
細胞質不親和性Cytoplasmic incompatibility (CI)
DNA腺嘌呤甲基轉移酶DNA adenine methyl transferase (Dam)
修改Modification (Mod)
甲基轉移酶Methyl transferase (Met)
pACYC-甲基轉移酶pACYC-methyl transferase (pACYC-met)
pET-調控轉錄因子pET-transcription factor (pET-TF)
pGEMT-甲基轉移酶pGEMT-methyl transferase (pGEMT-met)
搶救Rescue (Res)
米象甲基轉移酶 Sitophilus oryzae (Sli_met)
沃巴赫氏體 Wolbachia pipients (Wolbachia)
沃巴赫氏體表面蛋白 Wolbachia surface protein (Wsp)

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