A-超家族芋螺毒素基因的克隆及内含子遗传进化分析  

Cloning A-superfamily conotoxin genes and phylogenetic analysis of their introns

作　　者：孟海玲[1] 庞静[1] 林波[1] 李庆[1] 李明祥[1] 马艺珍[1] 罗素兰[1] 长孙东亭[1] 

机构地区：[1]海南大学热带生物资源教育部重点实验室,海口市海洋药物重点实验室,海南海口570228

出　　处：《中国海洋药物》2015年第3期14-22,共9页Chinese Journal of Marine Drugs

基　　金：国家自然科学基金项目(41366002;81160503);国家国际科技合作专项(2011DFR31210);海南省社会发展科技专项(SF201328);海口市重点科技计划项目(2013-16);长江学者和创新团队发展计划(IRT1123)资助

摘　　要：目的从中国南海独特芋螺中克隆新的A-超家族芋螺毒素基因,并分析其内含子的遗传进化关系。方法以独特芋螺基因组DNA为模板,根据A-超家族芋螺毒素基因的信号肽保守序列和3'-非翻译区(3'-UTR)保守序列设计特异性引物,通过PCR方法扩增出目的基因片段,并将其连接到pMDTM19-T载体,转化到DH5α感受态细胞并测序,同时对新的A-超家族芋螺毒素基因内含子进行遗传进化分析。结果从海南产独特芋螺基因组DNA中克隆到4条新的完整A-超家族芋螺毒素基因。CaI-M1a,CaI-M1b,CaI-M1c是编码同一种芋螺毒素CaI-M1的3个基因,它们的内含子序列存在较大的差异,特别是其中的GT重复不同。CaXXVII-M2编码产生的成熟肽含有5个半胱氨酸,与传统的属于A-超家族的α-芋螺毒素含有4个半胱氨酸的模式不同。结论首次在独特芋螺中克隆到4个新的含有完整内含子的A-超家族芋螺毒素基因,并表明其内含子的多样性与物种进化和捕食习性有一定的联系。Objective To clone novel A-superfamily conotoxin genes fromConus caracteristicus native to Hainan,and analyze phylogeny of their introns.Methods The C.caracteristicus Genomic DNA was used as template for gene cloning of novel A-superfamily conotoxins using the conserved DNA sequences of signal peptide and 3＇-UTR（untranslated region）of A-superfamily genes by PCR.The targeted DNA was ligated into the pMDTM19-T vector and transformed into E.coli DH5（competent cell.Then the individual bacterial colonies were individually sequenced.Finally,gene structure,sequence characteristics and phylogeny of their introns were analyzed.Results 4A-superfamily conotoxins were cloned from genomic DNA of C.caracteristicus native to Hainan,China.CaI-M1 a,CaI-M1 b CaIM1 cencoded the same conotoxin CaI-M1 but the sequences of their introns were different in the number of GT repeats.And CaXXVII-M2 encoded a new member of A-superfamily with five-Cys framework（CC-C-C-C）,which did not belong to classific（-conotoxins（CC-C-C）.ConclusionIt was the first time to clone four novel A-superfamily conotoxin genes with complete introns fromC.caracteristicus.Furthermore,the phylogenetic tree of A-superfamily conotoxin introns showed that introns was associated with conus species and their prey.

关 键 词：A-超家族芋螺毒素基因 基因克隆 内含子 遗传进化树 

分 类 号：R931[医药卫生—生药学]