基于转录组测序的马尔尼菲青霉菌实时荧光定量PCR内参基因筛选与鉴定  

Selection and Idetification of Reference Genes for qRT-PCR in Penicillium marneffei based on RNA-Seq

作　　者：陶玉婷[1] 卢姗[1] 王超[1] 王俊杰[1] 康雪晴 蓝秀万[1,2,3,4] 梁晓乐[2] 梁莹[3,5] 何志义[3,6] 

机构地区：[1]广西医科大学基础医学院生物化学与分子生物学教研室,南宁530021 [2]广西医科大学基础医学中心实验室,南宁530021 [3]广西高校基础医学研究重点实验室,南宁530021 [4]广西高校生物分子医学研究重点实验室,南宁530021 [5]广西医科大学基础医学院微生物学教研室,南宁530021 [6]广西医科大学第一附属医院呼吸科,南宁530021

出　　处：《基因组学与应用生物学》2016年第5期1042-1048,共7页Genomics and Applied Biology

基　　金：国家自然科学基金项目(No.81160195)资助

摘　　要：马尔尼菲青霉菌是一种温度依赖性双相型条件致病真菌,可感染免疫缺陷人群,区域流行于东南亚地和我国南方,但其双相转换和致病性分子机制尚不清楚。实时荧光定量PCR是马尔尼菲青霉菌双相转换和致病性分子机制研究的重要手段,但仍缺乏用于马尔尼菲青霉菌实时荧光定量PCR标准化分析理想的内参基因。本研究根据转录组测序数据筛选了四个表达最稳定的Pfp、Rp123、FacpA、DigA基因与传统的内参基因18Sr RNA、β-actin(Act1)、β-tubulin(Btu)作为候选内参基因,用Best Keeper和ge Norm软件进行基因表达稳定性分析。结果表明FacpA和DigA基因在马尔尼菲青霉菌双相转换过程中表达最为稳定,可作为用于马尔尼菲青霉菌实时荧光定量PCR标准化分析的内参基因。Penicil lium marneffei is a temperature-dependent dimorphic opportunity pathogenic fungi, causing lethal systemic infections in immunocompromised population which is endemic in Southeast Asia and southern China. The molecular mechanisms of dimorphic phase transition and pathogenicity underlying are still unclear.To explore the molecular mechanisms, quantitative real time PCR（qRT-PCR） has been one of the most important approaches to perform gene expression analysis. However, more suitable reference genes for q RT-PCR data normalization need to be developed. In this study, we selected 4 reference genes（Pfp, Rp123, FacpA, DigA）,expressing stably during dimorphic phase transition from our previous RNA-seq dataset and 3 traditional reference genes（18S rRNA, Act1, Btu） as candidate reference genes. Two analytical software packages（Best Keeper and ge Norm） were used to assess the stability of the expression level of these genes. The results showed that FacpA and DigA were the optimum pair of reference ge nes at all dimorphic phase transition stages and intwo different strains and were recommended for use as the endogenous control for gene expression analysis in P. marneffei by qRT-PCR.

关 键 词：马尔尼菲青霉菌 内参基因 实时荧光定量PCR DigA基因 FacpA基因 

分 类 号：R379[医药卫生—病原生物学]