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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;魚腥藻;RNA polymerase;in vitro transcription;RNA聚合■;生體外轉錄
出版社: 植物學系
研究利用polyethyleneimine及ammonium sulfate 等蛋白純化步驟,自臺
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
均有轉錄的能力,而且轉錄的活性會受 rifampicin 抑制。利用聚合■連
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
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,
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

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.
Appears in Collections:生命科學系所

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