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

Real-time RT-PCR based on DNA subtraction for absolute quantification of gene expression in engineered lactic acid bacteria

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CBHⅡ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。

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.