Acta Agrestia Sinica

Acta Agrestia Sinica ›› 2023, Vol. 31 ›› Issue (4): 1001-1007.DOI: 10.11733/j.issn.1007-0435.2023.04.008

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Screening of Internal Reference Genes of Bupleurum marginatum var. stenophyllum and Analysis of Gene Expression of Saikosaponin Key Enzymes

SUN Zhe1,2, DU Xiao-rong1,2, HUA Long1, LI Ao-xuan1,2, SONG Yun1,2, HAN Ming-zhe1, QIAO Yong-gang1,2   

  1. 1. College of Life Sciences, Shanxi Agricultural University, Taigu, Shanxi Province 030801, China;
    2. Shanxi key lab. for modernization of TCVM, Taigu, Shanxi Province 030801, China
  • Received:2022-10-18 Revised:2022-11-21 Published:2023-04-28

窄竹叶柴胡内参基因筛选及皂苷合成关键酶基因表达分析

孙哲1,2, 杜晓蓉1,2, 花龙1, 李澳旋1,2, 宋芸1,2, 韩明哲1, 乔永刚1,2   

  1. 1. 山西农业大学生命科学学院, 山西 太谷 030801;
    2. 中兽医药现代化山西省重点实验室, 山西 太谷 030801
  • 通讯作者: 乔永刚,E-mail:sxndqyg@126.com
  • 作者简介:孙哲(1997-),男,汉族,山西河津人,硕士研究生,主要从事药用植物遗传学相关研究,E-mail:297093122@qq.com
  • 基金资助:
    国家重点研发计划(2019YFC1710801)资助

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Abstract: Bupleurum marginatum Wall. ex DC. var. stenophyllum (Wolff) Shan et Y. Li is a perennial shrubby herb of the genus Bupleurum,with high medicinal value as the root is known as Tibetan Bupleurum. In this experiment,real-time quantitative PCR technology was used,combined with the three different data analysis softwares of the geNorm,NormFinder and BestKeeper to screen out the genes that can be stably expressed in different organs of B. marginatum var. stenophyllum from the five commonly used candidate genes:Actin,α-tubulin,β-tubulin,Cyclophilin and EF-1α as the internal reference genes,and then the selected internal reference genes were used to analyze the key enzyme genes of saikosaponin by the relative expression levels of HMGR,IPPI,FPS,SS,β-AS in different organs of B. marginatum var. stenophyllum. The results showed that β-tubulin gene could be used as an internal reference gene for its stable expression in different organs of B. marginatum var. stenophyllum;different saikosaponin synthase genes were expressed differently in different organs of B. marginatum var. stenophyllum. Except for IPPI gene which had the highest expression in seeds. The expression levels of HMGR,FPS,SS and β-AS in roots were higher than those in other organs,and the expression levels of HMGR,FPS and SS in lateral roots were higher than those in main roots. Therefore,the expression levels of key enzyme genes for saponin synthesis in different parts of B. marginatum var. stenophyllum were different. The optimal internal reference gene is β-tubulin gene,which can provide a basis for molecular biology research of B. marginatum var. stenophyllum and this research could promote its rational development and wide utilization.

Key words: Bupleurum marginatum var. stenophyllum, Internal reference gene, Saikosaponin, Real-time quantitative PCR, Expression analysis

摘要： 窄竹叶柴胡(Bupleurum marginatum var. stenophyllum)为柴胡属多年生高大型草本植物，以根入药称为藏柴胡，具有较高的药用价值。本试验采用实时荧光定量PCR技术，利用geNorm、NormFinder及BestKeeper3种不同数据分析软件，从5个常用的候选基因(Actin,α-tubulin,β-tubulin,Cyclophilin,EF-1α)筛选出可在窄竹叶柴胡不同器官稳定表达的基因作为内参基因，并利用筛选得到的内参基因分析皂苷合成关键酶基因(HMGR,IPPI,FPS,SS,β-AS)在窄竹叶柴胡不同器官的相对表达量。结果表明β-tubulin基因可作为窄竹叶柴胡不同器官稳定表达的内参基因；皂苷合成关键酶基因在窄竹叶柴胡的不同器官中表达量有显著差异，IPPI基因在种子中表达量最高，其他基因在根中的表达量均高于在其他器官中的表达量，其中HMGR,FPS,SS在侧根的表达量高于主根。因此，窄竹叶柴胡各器官皂苷合成关键酶基因的表达量不同，最适内参基因为β-tubulin基因，本研究为窄竹叶柴胡分子生物学研究提供一定依据，促进其合理开发和利用。

关键词: 窄竹叶柴胡, 内参基因, 柴胡皂苷, 实时荧光定量PCR, 表达分析

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