水稻黑条矮缩病毒和水稻条纹叶枯病毒侵染下水稻qRT-PCR内参基因的筛选

Reference gene selection for real-time fluorescence quantitative PCR analysis in rice plants infected by Rice black-streaked dwarf virus or Rice stripe virus

病毒侵染通常会干扰寄主细胞的生理代谢过程,分析病毒侵染后寄主基因的表达差异,将为病毒与寄主之间的互作研究提供重要的分子基础。实时荧光定量PCR(qRT-PCR)是目前基因表达分析中应用最广泛的方法之一,选择合适的内参基因对实验进行校正和标准化至关重要。但是,一些常用作内参的看家基因会受到病毒侵染的影响。本研究中,以感染水稻黑条矮缩病毒(Rice black-streaked dwarf virus,RBSDV)和水稻条纹叶枯病毒(Rice stripe virus,RSV)的水稻总RNA为材料,利用qRT-PCR技术和3个统计学软件探讨了10个常用内参基因在病毒侵染下的稳定性。结果显示,感染RBSDV和RSV水稻中表达最稳定的都是UBC和β-TUB。因此,可选用UBC和β-TUB组合作为分析RBSDV和RSV侵染过程的水稻内参基因。

Virus infection disrupts the normal metabolism and biological processes of the host. At the molecular level, virus infection triggers a global change in host gene expressions, in particular, the expression changes of host genes that are associated with the stress and defense against pathogen attacks. Thus, studying the alteration of plant gene expression upon virus infection may provide detailed insights into the molecular interaction between virus and host. The real-time fluorescence quantitative PCR （qRT-PCR） has been widely used to investigate the changes in the cellular gene expression. For the accuracy of qRT-PCR analysis, normalization using the appropriate internal control genes is necessary. However, sometimes the expression of housekeeping genes that are commonly used as internal controls is affected by virus infection. In this study, the effect of Rice black-streaked dwarf virus （RBSDV） or Rice stripe virus （RSV） infection on the transcript expression levels of 10 rice （Oryza sativa） housekeeping genes including 18S, 25S, ACT, β-TUB, eEF-1α, eIF-4a, GAPDH, UBC, UBQ-5 and UBQ-10 were assessed by qRT-PCR and then further analyzed by three commonly used algorithms： geNorm, NormFinder and BestKeeper. The qRT-PCR results, combined with three algorithms analyses, revealed that half of the transcript levels of internal control rice genes were affected by RBSDV infection while most of the genes were stable in RSV-infected rice plants. Among those genes, UBC and β-TUB were the reference genes with the most unaltered transcript levels by infection with RBSDV and RSV. Taken together, our results showed that RBSDV and RSV infections differently affected the transcriptional expression of housekeeping genes that were commonly used for internal control in qRT-PCR. Thus, determining the suitable internal reference genes is crucial for profiling the alteration of gene expression pattern by different virus infections.