无患子RT-qPCR内参基因的筛选与验证  

Reference Genes Selection and Validation for RT-qPCR in Sapindus mukorossi

作　　者：徐圆圆 赵国春 郝颖颖 翁学煌 陈仲 贾黎明[1,2,3] XU Yuan-yuan;ZHAO Guo-chun;HAO Ying-ying;WENG Xue-huang;CHEN Zhong;JIA Li-ming(Key Laboratory of Silviculture and Conservation of the Ministry of Education,College of Forestry,Beijing Forestry University,Beijing 100083;National Energy R&D Center for Non-food Biomass,Beijing Forestry University,Beijing 100083;National Innovation Alliance of Sapindus Industry,Beijing Forestry University,Beijing 100083;Yuanhua Forestry Biological Technology Co.,Ltd.,Sanming 354500;Beijing Advanced Innovation Center for Tree Breeding by Molecular Design,Beijing Forestry University,Beijing 100083)

机构地区：[1]北京林业大学林学院省部共建森林培育与保护教育部重点实验室,北京100083 [2]北京林业大学国家能源非粮生物质原料研发中心,北京100083 [3]北京林业大学无患子产业国家创新联盟,北京100083 [4]福建源华林业生物科技有限公司,三明354500 [5]北京林业大学林木分子设计育种高精尖创新中心,北京100083

出　　处：《生物技术通报》2022年第10期80-89,共10页Biotechnology Bulletin

基　　金：国家自然科学基金项目(32071793);国家科技基础资源调查专项(2019FY100803)。

摘　　要：无患子根、茎、叶、花和果皮均含有生物活性物质三萜皂苷,为了解三萜皂苷生物合成途径中相关基因的表达水平,需要筛选稳定表达的内参基因。以无患子根、茎、叶、芽、雄花、雌花和不同发育时期的果皮为材料,根据无患子转录组数据,选择Sm18S,SmACT,SmTBCC,SmEF-1α,SmRPL1,SmRPS26,SmUBC12,SmUBP等8个基因作为候选内参基因。通过实时荧光定量PCR(RT-qPCR)检测这些候选内参基因的表达量,并利用geNorm、NormFider和BestKeeper三个软件及RefFinder在线分析工具评价候选内参基因的稳定性。结果表明,8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;geNorm、NormFinder和BestKeeper 3个软件筛选出的最佳内参基因略有不同;综合分析结果表明SmACT、SmRPL1和SmUBP表达较为稳定,SmEF-1α稳定性最差;以SmACT、SmACT+SmRPL1组合和SmACT+SmRPL1+SmUBP组合为内参对三萜皂苷生物合成途径中的8个候选基因进行校准所得的表达量基本上保持一致,且相对表达量结果与转录组数据基本保持一致,表明SmACT、SmACT+SmRPL1组合和SmACT+SmRPL1+SmUBP组合可作为无患子三萜皂苷生物合成途径相关基因表达研究的内参基因,同时也可以为无患子及近缘植物的其他生物学过程中的基因表达研究提供参考。The roots,stems,leaves,flowers,and pericarps of Sapindus mukorossi Gaertn.are rich in bioactive triterpenoid saponin.In order to understand the expressions of related genes in triterpenoid saponin biosynthesis pathway,it is necessary to screen the stablyexpressed reference genes.In this study,8 traditional reference genes including 18S rRNA(guanine1575-N7)-methyltransferase(Sm18S),actin-related protein 8(SmACT),elongation factor 1-alpha(SmEF-1α),large subunit ribosomal protein L1(SmRPL1),small subunit ribosomal protein S26e(SmRPS26),tubulin-specific chaperone C(SmTBCC),ubiquitin-conjugating enzyme E2 M(SmUBC12),E3 ubiquitin-protein ligase BAH(SmUBP)were selected as candidate reference genes based on the RNA-seq data of roots,stems,leaves,buds,male flowers,female flowers,and developing pericarps of S.mukorossi.The expressions of these candidate reference genes were detected by RT-qPCR in the root,stem,leaf,bud,male flower,female flower,and pericarps at different developmental stages,and the stability of them were then evaluated by geNorm,NormFinder and BestKeeper software and RefFinder online analysis tool.The results showed that the expressions of 8 candidate reference genes differed in the variations among all samples.And the optimal reference genes screened by geNorm,NormFinder and BestKeeper were also slightly different.The results of comprehensive analysis showed that SmACT,SmRPL1 and SmUBP expressed quite stably,while the stability of SmEF-1αwas the lowest among all candidate reference genes.The RT-qPCR results of 8 triterpenoid saponin biosynthesis related genes using SmACT,SmACT+SmRPL1 and SmACT+SmRPL1+SmUBP as the reference genes respectively were in accordance with the results of RNA-seq.Thus,it is concluded that SmACT and the reference gene combinations of SmACT+SmRPL1 or SmACT+SmRPL1+SmUBP can be used as reference genes for gene expression analysis of triterpenoid saponin biosynthesis pathway in S.mukorossi and other biological processes in S.mukorossi and related plants.

关 键 词：无患子 内参基因 实时荧光定量PCR 三萜皂苷生物合成 表达分析 

分 类 号：Q943.2[生物学—植物学] S792.99[农业科学—林木遗传育种]