基于DNA扣除法的Real-timeRT-PCR对工程乳酸菌外源基因.pdfVIP

生物工程学报 Chin J Biotech 2009, August 25; 25(8): 1240- journals . Chinese Journal of Biotechnology ISSN 1000-3061 cjb@ © 2009 Institute of Microbiology, CAS CSM, All rights reserved 生物技术与方法 基于DNA 扣除法的Real-time RT-PCR 对工程乳酸菌外 源基因表达的绝对定量分析 史瑞, 刘飞, 霍贵成, 杨丽杰 东北农业大学 乳品科学教育部重点实验室, 哈尔滨 150030 摘 要: 本研究旨在利用Real-time RT-PCR对外源基因在工程乳酸菌中的表达进行定量分析, 建立一种新的Real-time RT-PCR分析方法。采用玻璃珠热酚法提取工程乳酸菌总RNA, 对外源目的基因的反转录(含有cDNA和DNA)样品和非反 转录(仅含DNA)样品进行Real-time PCR检测, 根据经典绝对定量方法并结合DNA 扣除法进行分析, 将得到的Ct值通过 标准曲线换算为样品拷贝数, 通过从反转录样品中扣除DNA样品的拷贝数的量, 去除了DNA对实验结果的影响, 得出 最终的定量结果。采用以上方法分析工程乳酸乳球菌NZ9000 中外源纤维素酶基因CBH Ⅱ的表达情况, 对表达量较低的 目的基因进行转录水平的分析, 避免了RNA的损失, 得到了外源基因表达的量为(1.28 ±0.02)×101 copies/cfu 。这种基于 DNA扣除法的Real-time RT-PCR绝对定量方法可以有效地对外源基因在工程乳酸菌中的表达进行分析。 关键词: 乳酸菌, Real-time RT-PCR, 绝对定量, 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 bacte