短小芽孢杆菌实时荧光定量PCR分析中内参基因的筛选

doi:10.13560/j.cnki.biotech.bull.1985.2016.11.028

摘要/Abstract

摘要： 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。

关键词: 短小芽孢杆菌, 实时荧光定量PCR, 内参基因, geNorm, NormFinder

Abstract: In order to study the gene expression in Bacillus pumilus through real-time fluorescence quantitative PCR,we need to determine the appropriate reference gene firstly. The expressions of five candidate reference genes（16S rRNA,mecA,cadR,rpoB,andsphP）under different fermentation phases of B. pumilus were analyzed by real-time fluorescence quantitative PCR,and their expression stabilities were evaluated by geNorm and NormFinder software. According to the analysis by geNorm software,16S rRNA and mecA were the most stable genes and the number of optimal reference genes were 2. Analysis by NormFinder software revealed that mecA was the most stable gene. The differential expressions of 3 functional genes of B. pumilus indicated that 16S rRNA and mecA were appropriate reference genes,moreover,expression abundance of mecA 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.

Key words: Bacillus pumilus, real-time fluorescence quantitative PCR, reference gene, geNorm, NormFinder

贺婷停, 宋婷, 王超, 张长斌, 王海燕. 短小芽孢杆菌实时荧光定量PCR分析中内参基因的筛选[J]. 生物技术通报, 2016, 32(11): 99-106.

HE Ting-ting, SONG Ting, WANG Chao, ZHANG Chang-bin, WANG Hai-yan. Screening of Reference Genes in Bacillus pumilus by Real-time Fluorescence Quantitative PCR[J]. Biotechnology Bulletin, 2016, 32(11): 99-106.

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