北柴胡柴胡皂苷合成相关基因的分子克隆与组织表达分析
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摘要
北柴胡(Bupleurum chinense DC.)为伞形科柴胡属植物,是药用柴胡的主要来源之一,其中所含的主要有效成分柴胡皂苷类是一种齐墩果烷类型的三萜皂苷。目前,国内外对于柴胡皂苷的生物合成研究还很不深入。本文采用改良的trizol方法,成功的提取了北柴胡总RNA,然后以提取的总RNA为模板,采用RT-PCR方法,首次从北柴胡中克隆得到HMGR、IPPI、FPS和β-AS基因—4个柴胡皂苷合成途径中的基因。大小分别为470bp、532bp、466bp、1349bp,分别编码157、177、155、449个氨基酸多肽。对4个基因进行一系列序列分析(NCBI在线BLAST,特征性保守域分析,酶切位点分析,southern杂交分析,分子系统进化树分析)后表明克隆得到了4个基因保守性最强的核心片断。采用RT-PCR方法,在北柴胡中首次克隆得到6个适于实时定量RT-PCR的管家基因片段,片段大小分别为101bp(18S rRNA)、96bp(Cyclophilin)、128bp(EF1α)、121bp(L2)、101bp(β-tubulin)、99bp(Actin)。实时定量RT-PCR试验后,通过C_T值定量分析和genorm软件分析18S rRNA、Cyclophilin、EF1α、L2、β-tubulin、Actin基因在北柴胡各器官中表达的稳定性,得出β-tubulin基因是最稳定表达的内参基因,表达最不稳定的是18S rRNA基因。以β-tubulin为内参照,采用实时定量RT-PCR方法扩增,并用2~(-ΔΔCT)相对定量数据分析方法定量HMGR、IPPI、FPS、β-AS基因表达数据,结果显示,在柴胡皂苷合成途径中离终产物最远的HMGR基因和离终产物最近的β-AS基因均在根中表达量最高,根可能为合成柴胡皂苷的主要器官。本试验通过对柴胡皂苷合成相关酶的表达研究证实了北柴胡的各器官都有合成柴胡皂苷的能力,4个皂苷合成相关基因在北柴胡根、茎、叶、花、果实中均有表达,其中IPPI在茎中表达量最高,花中表达量最低;FPS在花中表达量最高,叶子中表达量最低。
Bupleurum chinense DC.(umbelliferae,bupleurum) is one of the main sources of medicinal bupleurum.The main effective component saikosaponin is oleanane-type triterpene saponin.At present, there were little research on the saikosaponin biosynthesis,cDNA fragments,encoding HMGR,IPPI,FPS andβ-AS which were involved in saikosaponin biosynthesis pathway in B.chinense were amplified by RT-PCR with the total RNA as a template,cDNA fragments of HMGR,IPPI,FPS andβ-AS gene were 470bp,532bp,466bp,1349bp in length encoding 157,177,155,449 amino acids respectively.The results of 4 genes sequence analysis(including blast analysis,conservative domain analysis,enzyme digestion sites analysis,southern hybridization analysis,phylogeneic tree analysis) indicated that the most conservative core fragments of 4 genes were cloned,cDNA fragments of six housekeeping genes were cloned by RT-PCR for the first time,the cDNA fragments were 101bp(18S rRNA),96bp(Cyclophilin), 128bp(EF1α),121bp(L2),101bp(β-tubulin),99bp(Actin) in length respectively.After the real time RT-PCR,expression stability of 18S rRNA,Cyclophilin,EF1α,L2,β-tubulin,Actin were analyzed with C_T value and genorm,the result indicated that the most stable gene isβ-tubulin,on the contrary,is 18S rRNA.Using theβ-tubulin as the internal reference standard,HMGR、IPPI、FPS、β-AS gene were amplified by real time RT-PCR.The data were analysed by 2~(-ΔΔCT).HMGR gene far from end product andβ-AS gene nearby end product showed the highest expression level on root.Root was the main organ for saikosaponin biosynthesis probably.The result of tissue expression of saikosaponin biosynthesis-related genes showed that saikosaponin can be synthesized in every organs from B. chinense.Four of the saikosaponin biosynthesis-related genes were all expressed in roots,stems,leaves, flowers and fruits from B.chinense.The highest expression level of IPPI was in stems and the lowest was in flowers.The highest expression level of FPS was in flowers and the lowest was in leaves.
Bupleurum chinense DC.(umbelliferae,bupleurum) is one of the main sources of medicinal bupleurum.The main effective component saikosaponin is oleanane-type triterpene saponin.At present, there were little research on the saikosaponin biosynthesis,cDNA fragments,encoding HMGR,IPPI,FPS andβ-AS which were involved in saikosaponin biosynthesis pathway in B.chinense were amplified by RT-PCR with the total RNA as a template,cDNA fragments of HMGR,IPPI,FPS andβ-AS gene were 470bp,532bp,466bp,1349bp in length encoding 157,177,155,449 amino acids respectively.The results of 4 genes sequence analysis(including blast analysis,conservative domain analysis,enzyme digestion sites analysis,southern hybridization analysis,phylogeneic tree analysis) indicated that the most conservative core fragments of 4 genes were cloned,cDNA fragments of six housekeeping genes were cloned by RT-PCR for the first time,the cDNA fragments were 101bp(18S rRNA),96bp(Cyclophilin), 128bp(EF1α),121bp(L2),101bp(β-tubulin),99bp(Actin) in length respectively.After the real time RT-PCR,expression stability of 18S rRNA,Cyclophilin,EF1α,L2,β-tubulin,Actin were analyzed with C_T value and genorm,the result indicated that the most stable gene isβ-tubulin,on the contrary,is 18S rRNA.Using theβ-tubulin as the internal reference standard,HMGR、IPPI、FPS、β-AS gene were amplified by real time RT-PCR.The data were analysed by 2~(-ΔΔCT).HMGR gene far from end product andβ-AS gene nearby end product showed the highest expression level on root.Root was the main organ for saikosaponin biosynthesis probably.The result of tissue expression of saikosaponin biosynthesis-related genes showed that saikosaponin can be synthesized in every organs from B. chinense.Four of the saikosaponin biosynthesis-related genes were all expressed in roots,stems,leaves, flowers and fruits from B.chinense.The highest expression level of IPPI was in stems and the lowest was in flowers.The highest expression level of FPS was in flowers and the lowest was in leaves.
引文
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