大型溞(Daphnia magna)线粒体基因组的测定与序列分析

Complete mitochondrial genome sequence of Daphnia magna( Crustacea: Cladocera)from Huaihe in China

利用Long-PCR及普通PCR技术,结合引物步移法测序,经拼接、组装后获得中国淮河大型溞(Daphnia magna)线粒体基因组全序列,并对其基因组成、结构特点等进行初步分析.大型溞线粒体基因组全长为14948 bp,A、T、G、C各碱基含量分别为32.37%、34.73%、15.58%、17.31%,表现出明显的AT偏倚.13个蛋白质编码基因、22个t RNA基因、2个rRNA基因和1个D-loop区(控制区)的排列方式与典型的节肢动物线粒体基因组的排列方式略有不同.除COI基因以CTG、ATP8基因以GTG、ND3基因以ATC以及ND6基因以ATT为起始密码子以外,其余9个蛋白质编码基因均以ATG作为起始密码子;9个蛋白质编码基因具有典型的完全终止子TAG或TAA,COI、COII、ND4和ND5等基因采用不完全终止密码子T.基因组包含9个基因间隔区,共81 bp,长度1~62 bp;13个基因重叠区,共77 bp,重叠碱基数在1~27 bp之间,最大重叠在16 S rRNA和tRNA^(Val)基因之间.在预测的22个tRNA基因的二级结构中,发现只有tRNA^(Ser1)基因未能形成完整的二级结构,其他21个基因均可形成典型的三叶草结构.16 S rRNA和12 S rRNA基因长度分别为1373 bp和752 bp,D-loop区全长289 bp.本研究结果为探讨大型溞在溞属中的系统学地位及其与其他种间的系统发生关系等问题提供了数据资源.

The complete sequence of mitochondrial genome of Daphnia magna was determined using long PCR,normal PCR and primers walking approaches. The results showed that the entire mitochondrial genome of Daphnia magna is 14948 bp in size,in which the content of A,T,G,C is 32. 37%,34. 73%,15. 58%,17. 31%,respectively. The mitochondrial genome organization and gene order of Daphnia magna were consistent with that of Arthropod,which contains 13 protein-coding genes（ PCGs）,22 tRNA genes,2 ribosomal RNA genes（ large and small ribosomal RNAs）,and a control region. Except for COI gene using CTG,ATP8 with GTG,and ND3 with ATC,and ND6 with ATT as the initiation codon,and other 9 PCGs of the mt DNA in Daphnia magna started with the typical ATG codon. TAG and TAA were used in 9 PCGs as usual termination codons,except for COI,COII,ND4 and ND5 genes with incomplete termination codon（ T）. There are 9 intergenic spacer sequencer totaling 81 bp（1- 62 bp for each sequence） and 13 overlapping sequences totaling 77 bp（1- 27 bp for each sequence）,scattered throughout the genome,respectively,and the largest overlap（27 bp） region is located between16 S r RNA and tRNA^（Val） genes. The secondary structures of 22 tRNAs were predicted and it was found that tRNA^（Ser1） failed to form a complete secondary structure,others have typical leaf clover secondary structures. The length of16 S rR NA,12 S rR NA and D-loop were 1373 bp,752 bp and 289 bp,respectively.The results of this study provide some useful molecular data to clarify the systematical status of Daphnia,as well as its phylogenetic relationships with other species.