藉用螢光蛋白融合技術，來觀察鏈黴菌內端粒複製必要蛋白的作用

To construct fluorescent protein fused Tpc and Tac for in vivo study during replicaton

張鈞博(Jin-Bo Chang)

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(2011年)

https://doi.org/10.6841/NTUT.2011.00357

摘要

鏈黴菌是普遍存在於土壤中的格蘭氏陽性菌，具有線形染色體以及質體，末端會有末端蛋白與DNA 5’端形成共價鍵。鏈黴菌線形DNA複製是由起始點往兩端複製，最後在延滯股(lagging strand)會留下一段無法完成複製的單股DNA，Tap會辨識末端單股DNA，Tap會藉由蛋白質之間的交互作用將末端蛋白與DNA聚合酶帶至單股的DNA，利用末端蛋白作為引子，最後由DNA聚合酶將剩下的區域補齊。SCP1上也有類似Tpg與Tap的兩種蛋白，分別是Tpc與Tac，可能是利用相同的機制結合至末端DNA。
本實驗使用螢光蛋白與Tac以及Tpc在基因層次進行融合，希望藉由觀察螢光蛋白可以了解這些Tac、Tpc在不同細胞分化時期的分佈及運動狀況，最終希望可以藉由Tpc-螢光蛋白來追蹤線形質體在鏈黴菌中的分佈。經由螢光蛋白融合後的質體可以支持質體線形複製，證實其末端蛋白依然具有功能。將質體送至低自體螢光的菌株內觀察，可以得知其末端蛋白的分佈狀態，間接推論出質體的分佈狀況。由實驗結果得知，在鏈黴菌生長過程中紅色螢光(Tpc)與綠色螢光Tac)在不同的細胞分化階段，會有不同的分布狀態，在孢子階段均有紅色螢光(Tpc)與綠色螢光(Tac)的存在。未來可以藉由螢光蛋白的移動來推論鏈黴菌是如何藉由接合生殖將質體傳遞給其他鏈黴菌。

關鍵字

鏈黴菌 ； 線形質體 ； 末端蛋白 ； 末端伴隨蛋白 ； SCP1

並列摘要

Streptomyces are to gram-positive bacteria, they have linear Chromosomes and linear plasmids. Capped by terminal protein covalently bond to the 5’end of DNA. Replication of linear DNA is bidirectional form an internal origin, and replicate the end of DNA the lagging strand cannot be replicated completely, and would leave a single-stranded overhang at the 3’end. In typical Streptomyces chromosomes Tap(terminal associate protein) recognizes the single strand DNA, and lead Tpg(terminal protein) and DNA polymerase to perform patching synthesis using, Tpg as the primer. Linear plasmid SCP1 encodes a different terminal protein, Tpc(Terminal protein of SCP1), and an accompany protein Tac(terminal associate protein of SCP1), both of which are uniqued for end patching in SCP1.
In this study, I used fluorescent protein fused Tpc and Tac, observe the distribution of these protein at different stages of growth and different under the microscope. The geusion protein could support the replication of mini-SCP1 plasmid, prove the fusion. The mini-SCP1 linear plasmid were introduced into a autofluorescent strain S.coelicolor FM145, in Streptomyces cell cycle the fluorescent had different partition. Red(Tpc) and green(Tac) fluorescent can be observed in spore and sporulation, these fluorescent can overlay with DAPI. The fusion gene report system can be use to track the plasmid movement during Streptomyces conjugation.

並列關鍵字

SCP1 ； Tpg ； Tap ； Tac ； Tpc ； linear plasmid ； Streptomyces

參考文獻

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