短小芽孢杆菌实时荧光定量PCR分析中内参基因的筛选

Screening of Reference Genes in Bacillus pumilus by Real-time Fluorescence Quantitative PCR

为了利用荧光定量PCR方法分析短小芽孢杆菌的基因表达水平,需要首先确定适合该菌株的荧光定量PCR分析内参基因。以短小芽孢杆菌的16S rRNA、mecA、cadR、rpoB及sphP共5个基因作为候选内参基因,利用实时荧光定量PCR的方法分析这5个候选基因在短小芽孢杆菌发酵培养不同时间点的表达情况,再用geNorm和NormFinder软件评估它们的表达稳定性。结果显示,利用geNorm软件分析得出16S rRNA和mecA是表达最稳定的基因,最适内参基因数为2。NormFindr软件分析得出mecA为最稳定的基因。对短小芽孢杆菌3个功能基因的差异表达分析结果表明,16S rRNA和mecA都是合适的内参基因,而mecA基因由于表达丰度适中,更适合于作为内参基因研究结构基因的表达。为了得到更准确的差异表达结果,也可用两个基因同时进行校正。

In order to study the gene expression in Bacillus pumilus through real-time fluorescence quantitative PCR,we need todetermine the appropriate reference gene firstly. The expressions of five candidate reference genes（16S r RNA,mec A,cad R,rpo B,andsph P）under different fermentation phases of B. pumilus were analyzed by real-time fluorescence quantitative PCR,and their expressionstabilities were evaluated by ge Norm and Norm Finder software. According to the analysis by ge Norm software,16 S rRNA and mec A were themost stable genes and the number of optimal reference genes were 2. Analysis by NormFinder software revealed that mec A was the most stablegene. The differential expressions of 3 functional genes of B. pumilus indicated that 16 S rRNA and mec A were appropriate reference genes,moreover,expression abundance of mec A was moderate,thus it was more suitable to be used as a reference gene in studying structural gene.Additionally,2 reference genes can also be used for calibration in order to acquire more accurate results.