半定量RT-PCR检测运动发酵单胞菌中外源基因转录水平的研究

Research on Exogenous Gene mRNA Levels in Zymomonas mobilis by Semi-quantitative RT-PCR

摘要:

本研究运用半定量RT-PCR法检测运动发酵单胞菌重组菌中外源基因xylB的转录水平。提取野生型运动发酵单胞菌CP4及其2个重组菌的总RNA, 检测无DNA污染后定量至同一浓度、并反转录为cDNA。观测目的基因xylB和内标基因16S rRNA的PCR扩增曲线、并确定合适的循环数, 选用相同量的cDNA为模板, PCR检测各样本中xylB相对16S rRNA的转录水平。结果表明野生型菌株CP4中xylB基因没有转录, 而两株重组菌中皆有xylB的转录本, 且转录丰度基本一致, 酶活测定也进一步证实该基因在重组菌中有效表达。该方法可用于鉴定运动发酵单胞菌中特定基因的转录水平, 是一种快速有效的检测方法。

Abstract:

An effective RT-PCR method was developed to detect exogenous gene xylB transcript levels in Zymomonas mobilis CP4. Total RNAs without genomic DNA contamination were purified from wild-type and gene engineering strains, and were quantified to the same concentration. Then, cDNAs synthesis and PCR analysis of these samples were conducted by reverse transcription PCR. The optimal number of cycles was determined by observing amplification profile of target gene xylB and internal control gene 16s rRNA, and relative expression levels of xylB in various samples were analyzed by RT-PCR. The results indicated that the xylB transcript was not be detected in CP4, however that could be found in recombinant strains, in which xylB transcription abundance was similar. The enzyme assay furthermore confirmed that effective expression of the target gene. The method provided a useful and rapid tool for detecting transcript levels of target genes from various samples of Z. mobilis.