基于转录组测序的斑地锦内参基因筛选及验证

Selection and Verification of the Reference Genes of Euphorbia maculata Based on Transcriptome Sequencing

斑地锦(Euphorbia maculata)是一种常用中药材,其中黄酮类化合物槲皮素是其主要药效成分之一。目前斑地锦槲皮素生物合成基因研究鲜见报道,而实时荧光定量PCR(RT-qPCR)是基因研究的常用方法之一,但具有合适的内参基因是RT-qPCR相对定量的前提条件。为了获得适合的斑地锦内参基因,本研究基于转录组测序结果,筛选出斑地锦EmSE、EmUBC、EmGDI1、EmLSM12、EmGBP、EmSYP22、EmTRI1、EmRSZ21、EmRPL7以及课题组前期得到的EmUBQ共10个基因作为侯选内参基因。通过RT-qPCR检测10个候选内参基因在斑地锦营养生长期和生殖生长期的根、茎、叶及果中的表达情况,利用geNorm、NormFinder和BestKeeper软件分析候选基因的表达稳定性,通过几何平均值法对不同软件所得结果进行整合并明确综合排名,即EmGDI1>EmUBQ>EmSE>EmUBC>EmLSM12=EmGBP>EmTRI1>EmSYP22> EmRSZ21=EmRPL7。以EmGDI1为内参基因,对槲皮素生物合成过程中的一个关键酶基因EmC4H进行表达分析,EmC4H在不同生长期不同组织内的表达差异与转录组测序结果趋势基本一致。本研究开发的内参基因EmGDI1可作为斑地锦不同生长期基因组织特异性表达研究中适合的内参基因。

The flavonoid quercetin is one of the main active ingredients in Chinese medicinal herb,Euphorbia maculata.At present,there are few reports on quercetin biosynthesis gene,and real-time quantitative PCR(RT-qPCR) is one of the common methods in gene research.But it is a prerequisite for relative quantitative RT-qPCR to have suitable reference genes.In order to obtain a suitable reference gene,this experiment was based on the results of transcriptome sequencing,EmSE,EmUBC,EmGDI1,EmLSM12,EmGBP,EmSYP22,EmTRI1,EmRSZ21,EmRPL7 and EmUBQ obtained by our research group in the early stage were selected as candidate reference genes.RT-qPCR was used to detect the expression of the candidate reference genes in roots,stems,leaves and fruits of E.maculata during vegetative and reproduction stages.The expression stability of the candidate genes was analyzed using geNorm,NormFinder and BestKeeper software.The results of different software were integrated and ranked by the geometric mean method.The ranking was in the order EmGDI1>EmUBQ>EmSE>EmUBC>EmLSM12=EmGBP>EmTRI1>EmSYP22>EmRSZ21=EmRPL7.EmC4H,a key enzyme gene in quercetin biosynthesis,was analyzed using EmGDI1 as the reference gene.The difference of EmC4H expression in different tissues at different stages was consistent with the trend of transcriptome sequencing.The reference gene EmGDI1 developed in this study could be used as a suitable reference gene for tissue-specific expression studies of different stages of E.maculata.