温度与条锈病菌双胁迫下小麦内参基因的选择

Selection of reference genes in wheat stressed by temperature and Puccinia striiformis f. sp. tritici

选择合适的内参基因是采用实时定量PCR研究基因表达获得可信数据的关键。以小麦在条锈病菌及温度双因素胁迫为研究对象,采用实时荧光定量PCR评价了CDC、RLI、ACTIN和26S基因的表达情况,采用ge Norm和Norm Finder软件进行了比较分析。结果表明,小偃6号小麦在常温(15℃±1℃)未接种条锈病菌,以及接种条锈病菌并培育至花斑期分别在高温(20℃±1℃)、变温(20℃到15℃)条件处理中,基因RLI表达不稳定,基因ACTIN在常温条件下以及在条锈病菌及高温处理条件下的表达量相对稳定,但在变温条件下波动较大。表达最稳定的基因是26S,其次是CDC基因,它们可以作为小偃6号-CYR32-温度互作体系中基因表达实时荧光定量PCR研究的内参基因,26S和CDC是最佳内参基因组合。

It is the key to select proper reference genes for getting valid data to analyze gene expression by quantitative real-time PCR （qRT-PCR）. The cell division control protein （ CDC）, RNase L inhibitor-like protein （RL/）, ATP-dependent 26S proteasome regulatory subunit （26S） and actin （ACTIN） genes in wheat were used to evaluate their expression level during wheat was stressed by temperature or/and Puccinia striiformis f. sp. tritici （PST） through qRT-PCR. The data were analyzed with geNorm and NormFinder software. The results showed that RL/gene expressed unsteadily in wheat cultivar Xiaoyan6 treated with normal temperature （15℃±1℃）, as well as the wheat inoculated with PST treated at high temperature （20℃±1℃） and variable temperature （from 20℃ to 15℃ ） during initial symptom-expression stage. Gene ACTIN displayed a stable expression level at normal temperature treatment, and high temperature with PST treatment, but not in variable temperature. The 26S was the most stable gene under all treatment conditions, followed by CDC gene. Both of them could be served as the best reference genes for normalization of qRT-PCR data in Xiaoyan6-PST-temperature interacting system. They could also be used as the best gene combination for reference gene.