北柴胡β-香树脂醇合酶基因家族成员鉴定及功能验证  

Identification and functional characterization ofβ-amyrin synthase gene family in Bupleurum chinense

作　　者：毛艳萍 李玉婵[1] 杨玉萍 李欢 张翼冠 侯大斌[1,3] MAO Yan-ping;LI Yu-chan;YANG Yu-ping;LI Huan;ZHANG Yi-guan;HOU Da-bin(School of Life Science and Engineering,Southwest University of Science and Technology,Mianyang 621000,China;College of Life Science and Biotechnology,Mianyang Teachers’College,Mianyang 621000,China;Aba Prefecture Xike Technology Innovation Center for Daodi Medicinal Materials,Aba 624000,China;Sichuan Institute for Translational Chinese Medicine,Chengdu 610041,China)

机构地区：[1]西南科技大学生命科学与工程学院,四川绵阳621000 [2]绵阳师范学院生命科学与技术学院,四川绵阳621000 [3]阿坝州西科道地中药材产业技术创新中心,四川阿坝624000 [4]四川省中医院转化研究医学中心,四川成都610041

出　　处：《中草药》2023年第11期3647-3654,共8页Chinese Traditional and Herbal Drugs

基　　金：四川省“十四五”农作物育种攻关项目(2021YFYZ0012-13);芸释医疗科技创新基金资助项目(20zh0234)。

摘　　要：目的从转录组层面对北柴胡Bupleurum chinenseβ-香树脂醇合酶(β-amyrin synthase,β-AS)基因家族成员进行鉴定,并对其表达及功能进行分析验证,以期为解析柴胡皂苷的合成和调控提供基础。方法基于全长转录组数据挖掘北柴胡β-AS基因家族成员(BcBASs),利用在线软件对各基因进行生物信息学分析,利用大肠杆菌和烟草验证关键基因的功能。结果从北柴胡转录组数据中挖掘到不冗余的β-AS基因6个,可分为3个亚家族,它们都具有典型保守区域DCTAE、MWCYCR和QW。进化分析表明,BcBAS1、BcBAS2、BcBAS4、BcBAS5、BcBAS6可能为单功能β-AS基因,BcBAS3可能为多功能β-AS基因,表达分析表明各基因在根和叶中的表达模式有差异。在大肠杆菌中成功表达了BcBAS1可溶性重组蛋白,相对分子质量约87000,烟草瞬时表达表明BcBAS1编码蛋白具有β-AS功能,能促进β-香树脂醇的积累。结论对北柴胡中6个不冗余的β-AS基因家族成员进行了鉴定和生物信息学分析,获得了Bc BAS1可溶性重组蛋白,在烟草中验证了BcBAS1为有功能的β-AS,为柴胡皂苷合成调控机制和生物合成研究奠定了基础。Objective To conduct identification analysis of theβ-amyrin synthase(β-AS)gene family in Bupleurum chinense from the transcription level,and evaluate its expression and function for elucidating the biosynthetic pathways and regulatory mechanisms of saikosaponins(SSs).Methods Based on the transcriptomics data of B.chinense,theβ-AS genes family(BcBASs)in B.chinense were mined,a series of tools were used for bioinformatics analysis and expression analysis of each gene,the key genes were transformed into Escherichia coli and tobacco for the functional validation.Results A total of six BcBASs members(BcBAS1—BcBAS6)were identified in this study,they were divided into three groups,all of them contained conserved regions including DCTAE,MWCYCR and QW.Evolutionary analyses indicated that BcBAS1,BcBAS2,BcBAS4,BcBAS5 and BcBAS6 may be monofunctionalβ-AS,BcBAS3 may be a multifunctionalβ-AS.Analysis of gene expression patterns showed that BcBASs were specifically expressed in different tissues.BcBAS1 was successfully expressed as solubl recombinant proteins in E.coli BL21(DE3),approximately 8.7×104.The tobacco transient expression assay showed that BcBAS1 coded asβ-AS,which affected the accumulation level ofβ-amyrin.Conclusion The members ofβ-AS gene family in B.chinense were identified,analyzed and validated.BcBAS1 soluble recombinant protein was obtained,and BcBAS1 was verified as a functionalβ-AS in tobacco.The results of this study will provide an important foundation for revealing the biosynthetic pathways and regulatory mechanisms of SSs.

关 键 词：北柴胡 β-香树脂醇合酶 基因家族 表达分析 功能验证 

分 类 号：R286.12[医药卫生—中药学]