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研究生: 阮越康
Nguyen Viet Khang
論文名稱: 蘇雲金芽孢桿菌含有兩種Prolyl-tRNA synthetase基因
Bacillus thuringiensis possesses two prolyl-tRNA synthetase genes of distinct origins
指導教授: 王健家
Chien-Chia Wang
口試委員:
學位類別: 碩士
Master
系所名稱: 生醫理工學院 - 生命科學系
Department of Life Science
論文出版年: 2018
畢業學年度: 106
語文別: 英文
論文頁數: 55
中文關鍵詞: 蘇雲金芽孢桿菌基因水平轉移欖樹
外文關鍵詞: Prolyl-tRNA synthetase, Horizontal gene transfer, Olive
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  • Prolyl-tRNA synthetase (ProRS)是一種第二類型tRNA合成酶,按照序列分析兩種不同構造的ProRS存在於不同物種中:原核型與真核型。多數細菌只含有一種原核型ProRS基因,但蘇雲金芽孢桿菌含有兩種ProRS基因:proS1與proS2。令人驚奇的是,這兩種基因擁有不同的來源,且proS1的表現量遠大於proS2。根據同源tRNAPro的辨識部位,proS1應該是構造性基因,但proS2的功能則未知。這個多出來的proS2基因猜測是來自於基因水平轉移 (HGT),其所解碼的蛋白質可能在逆境中拯救proS1功能或參與含proline抗生素的合成。至於蘇雲金芽孢桿菌是如何將proline接到其對應tRNA上的機制,我們尚未能做完整解釋。ProRS的親源演化分析告訴我們,蘇雲金芽孢桿菌ProRS2和植物ProRS2有很高的序列同源性。另外,這細菌被發現和橄欖樹 (Olea europaea)有共生關係,這個發現暗示proS2基因可能是為了生存必須而從橄欖樹水平轉移過來的可能性。探討兩物種之間的生物相剋相生關係可以讓我們更加了解生物水平轉移的機制,也能對於ProRS的抑制機制獲得進一步的解釋。


    Prolyl-tRNA synthetase (ProRS) is a class II synthetase which, according to sequence analysis, occurs in various organisms with one of two distinct structural architectures: prokaryotic-like and eukaryotic-like. While most of bacteria contain only one prokaryotic-like ProRS gene, Bacillus thuringiensis possesses two ProRS genes: proS1 and proS2. Intriguingly, both genes have different origins in which the expression of proS1 is much higher than that of proS2. Based on cognate tRNAPro identity elements, proS1 is supposed to be constitutive, whereas the function of proS2 remains unclear. The additional proS2 gene is hypothesized to be acquired by horizontal gene transfer (HGT) whose encoded protein (ProRS2) probably plays essential roles in rescuing the function of proS1 during stress conditions, participating in the biosynthetic pathways of antibiotics containing proline moiety, maintaining faithful translation. The question of how B. thuringiensis ProRS2 charges proline to its cognate tRNA remains to be characterized. The phylogenetic analysis of ProRS sequences showed that B. thuringiensis ProRS2 is well-aligned with plant ProRSs. The bacterium, additionally, can live symbiotically with olive tree (Olea europaea), raising a possibility that proS2 was transferred horizontally from the host tree during evolution course due to the necessity for survival. Understanding the allelopathic relationship between the two species could gain insight not only into the mechanism of HGT, but also into the inhibition of ProRSs.

    中文摘要 i Abstract ii Acknowledgement iii Table of Contents iv List of Figures and Tables v Abbreviations vi 1. Introduction 1 1.1 ProRS family is divided into two evolutionarily distinct groups 1 1.2 Different ProRS types recognize different tRNAsPro 1 1.3 Bacillus thuringiensis possesses two paralogous ProRSs 2 2. Materials and methods 4 2.1 Strains and culture medium 4 2.2 Preparation and transformation of E. coli competent cells 5 2.3 Preparation and transformation of yeast competent cells 7 2.4 Construction of BtProRSs 8 2.5 Protein preparation 8 2.6 SDS-PAGE 9 2.7 Western blot 10 2.8 Purification of His6-tagged proteins 12 2.9 Reverse-transcription (RT)-PCR 14 3. Results 16 3.1 B. thuringiensis contains two paralogous ProRSs and one p-type tRNAPro 16 3.2 Expression levels of B. thuringiensis ProRS genes under stress conditions 17 3.3 A proposed evolutionary history of ProRS 18 3.4 Why does B. thuringiensis possess two ProRSs? 20 4. Discussion 22 5. Reference 25 Figures 29 Supplementary Table 41

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