舟山眼镜蛇线粒体基因组全序列分析

Complete Sequence Analysis of Mitochondrial Genome of Chinese Cobra

【目的】对2条舟山眼镜蛇的线粒体基因组全序列进行了测定和分析,并初步探讨其系统发育地位。【方法】结合PCR产物TA克隆和步移测序技术,基于除nad6外12个蛋白编码基因的合并序列及其翻译的氨基酸序列,以蜥蜴为外群,用Neighbor-Joining(NJ)法构建了7种蛇的系统发育树。【结果】两者在长度上仅相差2bp,基因含量和排序完全一致,共编码37个基因(2个rRNA、22个tRNA和13个蛋白),其中tRNAGln、tRNAAla、tRNAAsn、tRNACys、tRNATyr、tRNASer(UCN)、tRNAGlu、tRNAPro和nad6由轻(L-)链编码,其他均由重(H-)链编码。13个蛋白编码基因的排列次序和相对位置与其他蛇类完全一致,起始密码子除cox1使用GTG外,其余均为ATG;与此同时,除nad1/4L/5、cox1和atp8外,其他均为不完全的终止密码子TA或T。NJ树分析发现,眼镜蛇科与游蛇科亲缘关系最近,随后依次与蝰科和蟒科聚在一起,各分支的支持值均高达90%以上。【结论】本研究证实了基于全线粒体基因组合并序列进行蛇类物种系统发育研究是可行的。

Objective To determinate the complete nucleotide sequence of the mitochondrial genome of two Chinese cobra individuals and to analyze its phylogenetic position. Methods The mitochondrial genomes were determined by TA-cloning and primer-walking sequencing. With Abronia graminea as the control and on the basis of the combined 12 mitochondrial coding genes and their corresponding translated amino acid sequences from 7 snakes, the phylogenetie tree of the snakes was constructed with the Neighbor-Joining （ N J） method. Results The difference of the length of mitochondrial DNAs （mtDNAs） from the two Chinese cobra individuals is only 2 bp. The mtDNAs from the two Chinese cobra individuals contained 37 genes （2 rRNAs, 22 tRNAs and 13 protein- coding genes） and 2 non-coding control regions. Of 37 genes, tRNA^Gla , tRNA^Ala, tRNA^Asn, tRNA^Cys, tRNA^Tyr,tRNA^Ser（UCN）, tRNA^Glu, tRNA^Pro and had6 were coded by light （L-） strand, others were coded by heavy （H-） strand. All the 13 protein-coding genes used ATG as initiation codon except coxl （GTG）. The nadl/4L/5, coxland atp8 were with TAA as termination codon, while others had incomplete termination codons of T or TA. The sequences and related position of 13 protein-coding genes were as the same as those in the others of snake family. The constructed phylogenetic trees showed that elapidae were clustered with colubridae at first, and then followed by viperidae and pythonidae /boidae, all the bootstrap values being higher than 90%. Conclusion The phylogenetic analysis method based on the combined sequences from the whole mitochondrial genome is feasible for the research of phylogenetic position.