Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (10): 80-89.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1616

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

XU Yuan-yuan1,2,3(), ZHAO Guo-chun1,2,3, HAO Ying-ying1,2,3, WENG Xue-huang4, CHEN Zhong1,2,3,5(), JIA Li-ming1,2,3()   

  1. 1. Key Laboratory of Silviculture and Conservation of the Ministry of Education,College of Forestry,Beijing Forestry University,Beijing 100083
    2. National Energy R&D Center for Non-food Biomass,Beijing Forestry University,Beijing 100083
    3. National Innovation Alliance of Sapindus Industry,Beijing Forestry University,Beijing 100083
    4. Yuanhua Forestry Biological Technology Co.,Ltd.,Sanming 354500
    5. Beijing Advanced Innovation Center for Tree Breeding by Molecular Design,Beijing Forestry University,Beijing 100083
  • Received:2021-12-30 Online:2022-10-26 Published:2022-11-11
  • Contact: CHEN Zhong,JIA Li-ming E-mail:yuanyuanxu_2016@163.com;zhongchen@bjfu.edu.cn;jlm@bjfu.edu.cn

Abstract:

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 stably-expressed 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-),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 SmACTSmRPL1 and SmUBP expressed quite stably,while the stability of SmEF- was the lowest among all candidate reference genes. The RT-qPCR results of 8 triterpenoid saponin biosynthesis related genes using SmACTSmACT+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.

Key words: Sapindus mukorossi Gaertn., reference gene, quantitative real-time PCR, triterpenoid saponin biosynthesis, expression analysis