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標題: 台灣分離株魚腥藻CH1生體外轉錄系統的建立
An in vitro transcription system for the Taiwan isolated cyanobacterium Anabaena sp. strain CH1
作者: 柴御清
Chai, Yun-Chen
關鍵字: Anabaena
RNA polymerase
in vitro transcription
出版社: 植物學系
摘要: 研究利用polyethyleneimine及ammonium sulfate 等蛋白純化步驟,自臺 灣水稻田中分離到的魚腥藻CH1品系中,大量製備具有轉錄活性的蛋白萃 取液。進行生體外轉錄反應分析後,發現此魚腥藻蛋白萃取液可以辨認菠 菜葉綠體rbcL(ribulose-1.5-bisphosphate carboxylase/oxygenase large subunit)基因起動子,也可有效辨認大腸桿菌的終止子,但與葉綠 體RNA聚合■不同的是,魚腥藻RNA聚合■萃取液對環狀及線狀的DNA模板 均有轉錄的能力,而且轉錄的活性會受 rifampicin 抑制。利用聚合■連 鎖反應,分別篩選出魚腥藻CH1品系之rbcLS基因及其起動子。其起動子區 域的核酸序列與魚腥藻PCC 7120有94%的相似性。由南方墨點法得知,此 rbcLS基因在染色體中為單一基因。將CH1品系rbcLS基因起動子,殖入具有 終止子之載體中,做為DNA模板。並配合CH1蛋白萃取液,針對反應時間、 溫度、DNA含量、酵素量、NTPs所須之濃度及離子濃度,建立魚腥藻CH1品 系最佳生體外轉錄反應的轉錄系統。當以魚腥藻CH1 rbcLS基因起動子, 配合魚腥藻RNA聚合■萃取液進行生體外轉錄反應時,可轉錄出約780nt大 小的專一性RNA產物;而菠菜葉綠體 RNA聚合■萃取液,則無法辨認此基 因起動子。但當兩種RNA聚合■萃取液混合進行反應時,其轉錄效率和只 含單一魚腥藻RNA聚合■萃取液比較,有加成的情形。推測此兩種蛋白萃 取液在進行轉錄反應時,蛋白分子間有交互作用的產生。
The RNA polymerase crude extract was purified from the Taiwan isolated cyanobacterium Anabaena sp. CH1 with polyethyeneimine and ammonium sulfate precipitation. These proteins could recognize the spinach chloroplast rbcL gene promoter and E. coli thr. terminator in vitro transcription system. The crude extract could transcribe both supercoil and linear DNA template with transcription activity could be inhibited by Rifampicin. This was different from spinach chloroplast RNA polymerase. We could isolate Anabaena CH1 rbcLS gene and promoter region by PCR(polymerase chain reaction). The DNA sequence of promoter region was 94% homologous with the Anabaena PCC 7120. The result of southern blot demonstrated the rbcLS gene was single copy in Anabaena CH1 chromosome. The Anabaena CH1 rbcLS gene promoter region was cloned into the RTa vector which expressed transcription activity after in vitro transcription was characterized by time course; DNA concentration ;enzyme concentration ; nucleotide concentration ; temperation ; and ion concentration constructed the optimal condition of in vitro transcription by Anabaena CH1. The Anabaena CH1 rbcLS gene promoter could be transcribed the 780nt RNA produced by Anabaena CH1 crude extract but the spinach chloroplast RNA polymerase couldn't recognize this promoter sequence. The mixture of the two crude extract proteins the transcription efficiency is better than single Anabaena CH1 crude extract. The pridiction of the mixture two enzymes has interaction in vitro transcription system.
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