鏈黴菌線型質體SLP2接合傳遞的cis-acting element

Identification of a cis-acting element on Streptomyces linear plasmid SLP2 for conjugal transfer

薛國偉(Kuo-Wei Hsueh)

指導教授 ： 黃志宏

國立臺北科技大學/工程學院/生物科技研究所/碩士(2009年)

摘要

革蘭氏陽性的鏈黴菌和其他細菌的基因體結構不同，是除了有環形質體外，還具有線型染色體和線型質體，在這些線型質體DNA的5’末端具有末端蛋白﹝TP﹞以共價鍵結的形式結合其上。鏈黴菌部分的線型質體具有接合生殖的能力，除了能將自己本身的線型質體﹝Donor﹞利用接合的方式傳遞到不同株的鏈黴菌中﹝Recipient﹞，也可以幫助將線型的染色體帶到Recipient中。已知細菌接合生殖的機制以大腸桿菌﹝E.coli﹞的F質體研究最為詳細，但F質體的接合生殖機制無法套用在鏈黴菌的線型質體，將線型質體完整的傳遞到recipient中。
本研究是想先藉由找出鏈黴菌線型質體的接合傳遞起始點﹝ oriT ﹞及區域，希望有助於了解鏈黴菌線型質體接合傳遞的機制。因此我選用Streptomyces lividans中已被定序完成且與接合生殖相關基因也大致瞭解的線型質體SLP2，利用實驗室開發高頻率線型質體同源重組的方法，去找出SLP2接合傳遞的起始點及區域。將構築好的同源線型質體送到具有SLP2質體的鏈黴菌中，藉由在鏈黴菌中發生一次性同源重組，會產生兩種不同的線型質體，各帶有不同段的SLP2片段，我再去測試這些帶有不同SLP2片段的線型質體其接合傳遞能力，以檢測哪個重組質體帶有SLP2的oriT。利用此策略，我把SLP2 oriT可能所在的區域縮小到約2 kb的DNA片段，位於SLP217.9 kb至19.9 kb的位置上。

關鍵字

鏈黴菌 ； SLP2 ； 同源重組 ； 接合傳遞的起始點 (oriT)

並列摘要

The topology of Streptomyces genome is different from other bacteria, because it has not only circular plasmid, but also linear chromosome and plasmid. These linear DNA replicons carry covalent-bound terminal proteins ﹝TPs﹞ at their 5’ end of telomeric DNA. Some of the linear plasmids of Streptomyces have ability of conjugation, and sometimes they also promote transfer their linear chromosome into recipient. The current established mechanism of conjugation is mostly based on circular F plasmid in E. coli system, but this cannot be applied to linear plasmid of Streptomyces. The mechanism of conjugal transfer in Streptomyces is not clear, therefore finding the origin of conjugal transfer﹝oriT﹞ in linear plasmids may help to understand this mechanism. By a strategy of homologous recombination that we can generate different recombinant DNA carrying different parts of SLP2 DNA fragment in vivo. Then we tested the transfer ability of this recombinant DNA where SLP2 was present. A truncated SLP2 variant plasmid would be transferred to recipient if it contains oriT of SLP2. By this method, the oriT was narrowed to an internal 2 kb DNA fragment, locating at the coordinate of 17.9~19.9-kb of SLP2.

並列關鍵字

Streptomyces ； homologous recombination ； SLP2 ； oriT

參考文獻

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