菜豆壳球孢侵染芝麻过程中内参基因的筛选  

Screening of reference genes in sesame during Macrophomina phaseolina infection

作　　者：赵辉[1] 张春艳[1] 文艺[1] 刘玉霞[1] 刘新涛[1] 倪云霞[1] 王飞[1] 刘红彦[1] 

机构地区：[1]河南省农业科学院植物保护研究所农业部华北南部农作物有害生物综合治理重点实验室河南省农作物病虫害防治重点实验室,河南郑州450002

出　　处：《中国油料作物学报》2017年第3期393-398,419,共7页Chinese Journal of Oil Crop Sciences

基　　金：国家自然科学基金(31301631);农业部现代农业产业技术体系(CARS-15-1-05)

摘　　要：为选择适合菜豆壳球孢(Macrophomina phaseolina)侵染芝麻过程中实时荧光定量PCR分析的内参基因,11个基因作为候选内参基因。经过PCR扩增效率筛选,β-肌动蛋白(ACTB)、泛素连接酶(UBC)、α-微管蛋白(TUBA)、γ-微管蛋白(TUBC)、3-磷酸甘油醛脱氢酶基因(GAPDH)、核糖体蛋白S5(RPS5-a,RPS5-b)和内部转录间隔区(ITS)8个基因符合要求,可用于稳定度筛选。利用实时荧光定量PCR技术,检测了这8个候选基因在菜豆壳球孢侵染芝麻8h、16h、24h和32h的表达情况。经Ge Norm软件分析,ACTB、GAPDH和RPS5-b等3个基因表达较稳定。经过最适内参基因数目分析,在菜豆壳球孢侵染芝麻过程中基因定量表达分析时,选择ACTB、GAPDH和RPS5-b的多基因组合作为内参,能够更准确地校正定量结果。To find out the accurate reference genes for gene expression analysis of Macrophomina phaseolina by qRT-PCR,11 housekeeping genes were detected in the pathogenic process. According to PCR amplification efficiencies,8 housekeeping genes were retained and their expression stabilities were evaluated by computer algorithms Ge Norm. The 8 genes included ubiquitin-conjugating enzyme gene UBC,glyceraldehyde-3-phosphate dehydrogenase gene GAPDH,ribosomal protein S5 gene RPS5（as RPS5-a and RPS5-b）,α-tubulin gene TUBA,β-actin gene ACTB,γ-tubulin gene TUBC and internal transcribed spacer ITS. Using qRT-PCR,the expression stability of the 8 genes from sesame samples were investigated at 8 h,16 h,24 h and 32 h post M. phaseolina inoculation. Ge Norm analysis revealed that the most stable genes were ACTB,GAPDH and RPS5-b. They showed their housekeeping genes features,and could be used jointly as reference genes of M. phaseolina for plant-microb interaction analysis.

关 键 词：芝麻茎点枯病菌 QRT-PCR 内参基因 芝麻 

分 类 号：S432.44[农业科学—植物病理学] S565.3[农业科学—农业昆虫与害虫防治]