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

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.