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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 a new conotoxin A-superfamily from the genus Taro in the South China Sea and analyze the genetic evolution of its introns. Methods Based on the unique snail genomic DNA as a template, specific primers were designed according to the conserved sequence of the signal peptide of the conotoxin gene of A-superfamily and the conserved sequence of 3’-untranslated region (3’-UTR) The target gene fragment was ligated into pMDTM19-T vector, transformed into DH5α competent cells and sequenced. At the same time, a new genetic analysis of the gene conotoxin gene of A-superfamily was performed. Results Four new complete A-superfamily conotoxin genes were cloned from the genomic DNA from Hainan. CaI-M1a, CaI-M1b and CaI-M1c are the three genes encoding the same conotoxin CaI-M1. There are big differences in their intron sequences, especially in GT repeats. The mature peptide produced by the CaXXVII-M2 encoding contains 5 cysteines and differs from the conventional 4-cysteine-containing cones in the A-superfamily. Conclusions Four novel conotoxin A-superfamily conotoxin genes were cloned for the first time in the genus Taro, and its intron diversity was related to species evolution and predation habits.