栝楼实时荧光定量PCR内参基因的筛选与验证

Selection and Validation of Reference Genes for Quantitative Real-time PCR Analysis in Trichosanthes kirilowii Maxim

栝楼是葫芦科栝楼属雌雄异株植物,为了解雌雄株差异基因表达水平,需要筛选稳定表达的内参基因。本研究根据3个栝楼品种苗期雌雄株叶片的转录组测序结果,筛选出12个候选内参基因。通过荧光定量PCR(RT-qPCR)检测内参基因的表达量,并利用geNorm、NormFinder和BestKeeper 3个软件评价候选内参基因的稳定性。结果显示,12个候选内参基因的表达量在所有样本间的变化幅度存在差异,综合分析3个评估软件结果得到Actin和GAPDH基因在所有样品中的表达最为稳定。栝楼雌、雄株部分差异基因的RT-qPCR分析结果与转录组结果相吻合,说明Actin和GAPDH基因适合作为栝楼基因表达研究的内参基因。本研究筛选出的最稳定的内参基因将为栝楼后续的基因表达研究提供校准依据。

Trichosanthes kirilowii Maxim is a dioecious plant of Trichosanthes in Cucurbitaceae.In order to understand the expression level of the difference genes between male and female plants,it is necessary to select the reference genes with stable expression.12 candidate reference genes were selected from the transcriptome sequencing results of the leaves of male and female plants of three T.kirilowii varieties at seedling stage.The expression level was detected by quantitative real-time PCR(RT-qPCR),and the stability of candidate reference genes was evaluated by geNorm,NormFinder and BestKeeper software.The results showed that the variation of the expression levels of 12 candidate reference genes were different among the samples.A comprehensive analysis was conducted on the results of the three evaluation software,and the results showed that the expression level of Actin and GAPDH genes was the most stable in all samples.The results of RT-qPCR analysis of some differential genes between male and female T.kirilowii were consistent with the transcriptome results,indicating that Actin and GAPDH genes are the suitable internal reference genes for T.kirilowii.The most stable internal reference genes selected in this study will provide a basis for the following gene expression study in T.kirilowii.