文章摘要
A-超家族芋螺毒素基因的克隆及其内含子遗传进化分析
Cloning of A-superfamily Conotoxin Genes and Their Introns Phylogenetic Analysis
投稿时间:2014-11-06  修订日期:2015-03-19
DOI:
中文关键词: A-超家族芋螺毒素基因  基因克隆  内含子  遗传进化树
英文关键词: A-superfamily conotoxin gene  gene cloning  intron  phylogenetic tree
基金项目:国家自然科学基金项目(面上项目,重点项目,重大项目)
作者单位E-mail
孟海玲 海南大学热带生物资源教育部重点实验室 779631101@qq.com 
庞静 海南大学海洋学院  
林波 海南大学热带生物资源教育部重点实验室  
李庆 海南大学海洋学院  
李明祥 海南大学海洋学院  
马艺珍 海南大学海洋学院  
罗素兰 海南大学热带生物资源教育部重点实验室  
长孙东亭 海南大学热带生物资源教育部重点实验室 zhangsudt@163.com 
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中文摘要:
      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,扩增产物连接到pMD?19-T载体,并进行转化、测序。对获得的新基因及其内含子进行分析, 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:克隆到4条新的A-超家族芋螺毒素基因组基因,含有完整的信号肽、前肽、内含子、成熟肽序列各个部分。其中有 3条基因编码相同的前体肽氨基酸序列,但是它们的内含子序列却存在较大的差异,特别是其中的GT重复不同。另有1条基因编码产生的成熟肽却含有5个半胱氨酸,与传统的属于A-超家族的α-芋螺毒素的含有4个半胱氨酸模式不同。对新发现的内含子序列构建了遗传进化树。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组基因,其内含子的多样性与物种进化有一定的联系,且与捕食习性有关。
英文摘要:
      Objective: To clone novel A-superfamily conotoxin genes from Conus caracteristicus native to Hainan, and analyze phylogeny of their introns. Methods: Genomic DNA as template was extracted from C. caracteristicus venom duct. Target genes were amplified by PCR using specific primers, which corresponded to conserved DNA sequences of signal peptide and 3"-UTR (untranslated region) of A-superfamily genes. The specific PCR products were ligated into the pMD?19-T vector, then transformed and sequenced. Characteristics of novel genes and their introns" phylogeny were analyzed. Results: The full-length DNAs of 4 new A-superfamily conotoxin genes were obtained, which consisted of signal peptide, propeptide region, intron and mature peptide. Among them, three A-superfamily conotoxin genes encoded the same amino acid sequence of their precursors. But the sequence of introns and their GT repeats had significant differences. One of the A-superfamily conotoxin genes encoded mature peptide with five cysteine framework (CC-C-C-C), which was different from regular α-conotoxins with four cysteine framework (CC-C-C). The phylogenetic tree of new introns was constructed. Conlusion: This is the first time to clone four novel A-superfamily conotoxin genes with complete introns from C. caracteristicus. Furthermore, phylogeny of A-superfamily conotoxin introns were associated with species and their prey.
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