齿缘摄龟线粒体基因组全序列与系统进化

The Complete Mitochondrial Genome of Cyclemys dentata and Phylogenetic Relationships among 20 Species of Turtles

采用常规PCR扩增并测序获得了齿缘摄龟(Cyclemys dentata)线粒体DNA(mtDNA)全序列,并研究了其基因组结构特点;根据20种龟的线粒体基因组重链蛋白编码基因序列,分别利用最大简约法(MP)、最大似然法(ML)和贝叶斯法(Bayesian)构建系统进化树,探讨这些龟鳖物种之间的系统进化关系。结果显示,齿缘摄龟线粒体基因组全序列长为16489 bp(GenBank登录号为JX455823),A+T含量为61.51%,编码37个基因,包括13个蛋白质编码基因,2个rRNA,22个tRNA和1个控制区(Dloop),基因组成与其他龟鳖类动物相似;非编码区D-loop长973 bp,包含1个中央保守区(CD),2个扩展终止结合序列区(ETASs),3个保守盒(CSBs);构建的MP树、ML树和Bayesian树的拓扑结构相似,闭壳龟属7种龟聚为一枝,拟水龟属6种龟聚为一枝,齿缘摄龟与黑桥摄龟聚在一枝,3种进化树均支持这些龟鳖物种现有的分类学地位。

In this study,the mitochondrial（ mt） genome of Cyclemys dentata was firstly obtained; the relationship among 20 turtles was then analyzed based on mitochondrial DNA（ mtDNA）. According to the mtDNA sequence of C. atripons which had been published in GenBank（ No. EF067858）,sixteen PCR primers were designed to amplify the mtDNA sequence of the C. dentata（ Table 1）. These PCR products were inserted into the pMD-18 T cloning vectors respectively,the restructured vectors were then sequenced,and these sequencing fragments were spliced by Lasergene 7. 0 software. The structure and gene order of C. dentata mtDNA were determined by referring to C. atripons mtDNA,and using tRNAscan-SE 1. 21 to predicted tRNAs.Based on 20 turtles’ mtDNA heavy chain protein-coding genes sequences,three phylogenetic trees were constructed by Maximum parsimony（ MP）,Maximum-likelihood（ ML） and Bayesian methods using threeprograms,respectively. The results indicated that the total length of mtDNA of C. dentata was 16 489 bp（ GenBank No. JX455823）,similar to other turtles’ mitochondrial genome. It encoded 37 genes,consisting of13 protein-coding genes,2 ribosomal RNA genes（ 12 Sr RNA and 16 Sr RNA）,22 transfer RNA genes（ tRNAs） and a control region（ D-loop）（ Fig. 1,Table 2）. The bases composition of C. dentata mt genome was the same as that of C. atripons. Its A ＋ T content was 61. 51%,A ＋ T contents of 13 protein-coding genes were from 57. 89% to 67. 86%,and their GC contents were low（ Table 3）. The average length of 20 turtles’ mt genomes was 16 692. 5 bp,including 34. 1% A,27. 0% T,26. 0% C and 12. 9% G（ Table 4）. OLsequence was 36 bp in length; it could fold into a hairpin secondary structure which contained a 10 bp arm and an 8-base ring（ Fig. 2）. The D-loop of C. dentata mtDNA contained a central domain（ CD）,two extended termination associated sequences（ ETASs） and three conserved sequence blocks（ CSBs）（ Fig. 3）. From the Fig. 4,we knew that the MP,ML and Bayesian trees had analogical topologies,seven species of genus Cuora clustered into a branch,six species of genus Mauremys clustered into another branch,and the C. dentata clustered together with the C. atripons. The analysis results of three phylogenetic trees supported the existing classification of these turtles.