生物工程學報 25 August 2009, 25(8):1240～1246

基于DNA扣除法的Real-time RT-PCR對工程乳酸菌外源基因表達的絕對定量分析

史瑞, 劉飛, 霍貴成, 楊麗杰

東北農(nóng)業(yè)大學 乳品科學教育部重點實驗室, 哈爾濱 150030

摘  要: 本研究旨在利用Real-time RT-PCR對外源基因在工程乳酸菌中的表達進行定量分析, 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, 對外源目的基因的反轉錄(含有cDNA和DNA)樣品和非反轉錄(僅含DNA)樣品進行Real-time PCR檢測, 根據(jù)經(jīng)典絕對定量方法并結合DNA 扣除法進行分析, 將得到的Ct值通過標準曲線換算為樣品拷貝數(shù), 通過從反轉錄樣品中扣除DNA樣品的拷貝數(shù)的量, 去除了DNA對實驗結果的影響, 得出最終的定量結果,。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的表達情況, 對表達量較低的目的基因進行轉錄水平的分析, 避免了RNA的損失, 得到了外源基因表達的量為(1.28±0.02)×10 1 copies/cfu。這種基于DNA扣除法的Real-time RT-PCR絕對定量方法可以有效地對外源基因在工程乳酸菌中的表達進行分析,。

關鍵詞: 乳酸菌, Real-time RT-PCR, 絕對定量, 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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