生物工程學(xué)報(bào) 25 August 2009, 25(8):1240～1246
基于DNA扣除法的Real-time RT-PCR對(duì)工程乳酸菌外源基因表達(dá)的絕對(duì)定量分析
史瑞, 劉飛, 霍貴成, 楊麗杰
東北農(nóng)業(yè)大學(xué) 乳品科學(xué)教育部重點(diǎn)實(shí)驗(yàn)室, 哈爾濱 150030
摘 要: 本研究旨在利用Real-time RT-PCR對(duì)外源基因在工程乳酸菌中的表達(dá)進(jìn)行定量分析, 建立一種新的Real-time RT-PCR分析方法。采用玻璃珠熱酚法提取工程乳酸菌總RNA, 對(duì)外源目的基因的反轉(zhuǎn)錄(含有cDNA和DNA)樣品和非反轉(zhuǎn)錄(僅含DNA)樣品進(jìn)行Real-time PCR檢測(cè), 根據(jù)經(jīng)典絕對(duì)定量方法并結(jié)合DNA 扣除法進(jìn)行分析, 將得到的Ct值通過(guò)標(biāo)準(zhǔn)曲線換算為樣品拷貝數(shù), 通過(guò)從反轉(zhuǎn)錄樣品中扣除DNA樣品的拷貝數(shù)的量, 去除了DNA對(duì)實(shí)驗(yàn)結(jié)果的影響, 得出最終的定量結(jié)果,。采用以上方法分析工程乳酸乳球菌NZ9000中外源纖維素酶基因CBHⅡ的表達(dá)情況, 對(duì)表達(dá)量較低的目的基因進(jìn)行轉(zhuǎn)錄水平的分析, 避免了RNA的損失, 得到了外源基因表達(dá)的量為(1.28±0.02)×10 1 copies/cfu。這種基于DNA扣除法的Real-time RT-PCR絕對(duì)定量方法可以有效地對(duì)外源基因在工程乳酸菌中的表達(dá)進(jìn)行分析,。
關(guān)鍵詞: 乳酸菌, Real-time RT-PCR, 絕對(duì)定量, DNA扣除法
Real-time RT-PCR based on DNA subtraction for absolute quantification of gene expression in
engineered lactic acid bacteria
Rui Shi, Fei Liu, Guicheng Huo, and Lijie Yang
Key Laboratory of Dairy Science, Ministry of Education, Northeast Agricultural University, Harbin 150030, China
Abstract: To evaluate the absolute quantification of a target gene transcription in engineered lactic acid bacteria, we developed the Real-time RT-PCR based on DNA subtraction. We isolated the total RNA from the bacteria samples by glass bead, and then analyzed the Ct data of real-time RT-PCR by DNA subtraction assay. Using this method, we successfully estimated the expression level of CBHII gene in the strain of genetic engineered Lactococcus lactis. Since this method could avoid the mRNA copy number loss, it could be used to estimate the expression of other genes in lactic acid bacteria.
Keywords: lactic acid bacteria, Real-time RT-PCR, absolute quantification, DNA subtraction
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