鏈黴菌是一種土壤中的微生物，它具有線型的染色體以及環形或線型的可接合傳遞質體。現今較明瞭的細菌接合傳遞機制主要為大腸桿菌的環型F質體，以滾輪複製單向單股DNA進行接合傳遞，但這樣的傳遞機制無法直接套用在鏈黴菌線型DNA的傳遞機轉，而且目前對此類線型質體的接合傳遞機制也尚未清楚。已知鏈黴菌線型質體上有段clt (cis-acting-locus of transfer)序列，在接合傳遞時具有clt 序列的DNA片段能傳遞到接受株中。因此，找到鏈黴菌線型質體上的clt座落位置，將對了解線型質體接合傳遞的機制有所幫助。
目前已得知clt可能在SCP1中2.4 kb大小的片段上，且剔除前段2 kb的片段依舊不影響線型質體SCP1接合傳遞的效率，所以推斷clt就在後段的0.4 kb上，但整段2.4 kb都剔除時無法取得轉型株，因為後段約0.4 kb片段包含了kor基因的一部分，kor基因被剔除導致鏈黴菌的kil基因大量表現，使得菌體無法存活。為了繼續尋找最後0.4 kb中clt的確切位置，我先將完整的kor基因分別插入授予株與接受株的染色體DNA上，使其能穩定表現kor基因，接著將授予株中SCP1質體的kor基因剔除，並轉型帶有最後0.4 kb片段的微型線型質體並測試其接合傳遞的效率。然而剔除kor基因依舊不影響線型質體SCP1的接合傳遞效率，可是先前許嘉津將原本不具接合傳遞能力的微型質體嵌入2.4 kb clt的不同候選片段後，能具有接合傳遞的能力，所以推測最後的0.4 kb並不是SCP1最主要的clt。根據先前許嘉津的資料與這次的實驗結果，推測clt可能座落於SCP1的tra基因下游。

The soil bacteria of the genus Streptomyces contain a linear chromosome and circular or linear conjugal plasmid. Well-known, classical conjugation mechanism base on E.coli circular F-plasmid, involving a single-stranded DNA molecule transfer by rolling- circle replication. However, the mechanism is not applied to Streptomyces linear plasmid, which is not well known so far. The previous studies had shown the cis-acting-locus of transfer (clt) is required for plasmid transfer. Therefore, locating the clt region on Streptomyces linear plasmid will contribute to understand the linear plasmid conjugal transfer.
We had known the clt is probably located on the 2.4 kb size fragment of Streptomyces linear plasmid SCP1, and the transfer frequency does not influenced while the former 2 kb was deleted. Thus, we suppose the clt should be located on the rear 0.4 kb fragment. However, we could not get transformer when all the 2.4 kb was deleted, because the rear 0.4 kb contains a part of the kor gene. Knocking out the kor cause the kil gene overexpression, and the Streptomyces unable to survive. For further narrow down the clt region, I integrated one copy of kor to the chromosomes of donor and recipient respectively, drives them expressing kor constantly. And then, I knocked out the kor of SCP1, and transformed the small plasmid containing the rear 0.4 kb fragment to the donor, and exam the frequency of conjugal transfer. However, knock out the kor still not influencing the transfer frequency of SCP1, but previous experiment showed that non-transferable small plasmid become mobilizing after integrating clt candidate fragments, so we suppose the rear 0.4 kb is not the most important clt of SCP1. According to the previous data by Chia-Chin Hsu and my experiment, we guess the clt may locate on the down stream of the tra gene.

中文摘要 i
英文摘要 iii
誌謝 v
目錄 vi
表目錄 viii
圖目錄 ix
第一章 緒論 1
1.1細菌的接合傳遞 1
1.2鏈黴菌的簡介 4
1.3鏈黴菌的線型染色體與線型質體 6
1.4鏈黴菌的接合傳遞 8
1.5 Tra protein 與 kil-kor system 9
1.6鏈黴菌的 cis-acting locus of transfer (clt) 12
第二章 材料與方法 16
2.1菌種及質體 16
2.2藥品、酵素 19
2.3培養基及緩衝溶液 19
2.4菌種之儲存 19
2.5大腸桿菌之轉型 20
2.6大腸桿菌質體之分離與純化 20
2.7鏈黴菌原生質體(protoplast)的製備與轉型 20
2.8鏈黴菌基因組DNA的分離與純化 20
2.9限制酶酵素、T4系列酵素及各式kit的處理與使用 21
2.10聚合酶連鎖反應PCR(polymerase chain reaction) 21
2.11脈衝電場型膠體電泳(pulsed-field gel electrophoresis) 21
2.12南方點墨法(Southern blot hybridization) 21
2.13鏈黴菌質體接合傳遞效率的計算(recombination frequency by
linear plasmid conjugation) 22
2.14 REDIRECT technology 22
第三章 實驗結果 23
3.1將2.4 kb的conjugal cis-acting element繼續縮小其範圍 23
3.2剔除SCP1的SCP1.91與D片段重疊的部分 27
3.3剔除SCP1.91不影響SCP1的接合傳遞效率 40
第四章 討論 43
參考文獻 46
附錄
A. Media and buffer 53
B. E. coli Competent cell preparation and transformation 55
C. Plasmid isolation from E. coli 58
D. Preparation of Streptomyces protoplast and transformation 59
E. Isolation the total DNA of Streptomyces 60
F. Polymerase Chain Reaction 61
G. Pulsed-field gel electrophoresis 62
H. Southern hybridization 64
I. REDIRECT technology 68

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