麦穗鱼线粒体基因组序列测定及分析

SEQUENCE AND ANALYSIS OF COMPLETE MITOCHONDRIALGENOME OF PSEUDORASBORA PARVA

利用麦穗鱼Pseudorasbora parva和相关鱼类的部分线粒体基因序列,设计出2对长批引物和30对短批引物,采用基于长PCR的2次PCR扩增法测定并注释麦穗鱼线粒体基因组全序列。结果表明,麦穗鱼线粒体基因组长16600bp,A+T含量为58.9%,37个基因位置及组成与其它硬骨鱼一致,均由13个蛋白编码基因、22个tRNA、2个rRNA基因和1个控制区(D-loop)组成。其中L链仅含8个tRNA(Pro、T yr、Ser、Ala、Asn、Cys、Glu、Gln)及ND6基因,其余基因皆由H链编码。基因排列紧密,间隔序列共计13处64bp,长度从1～32bp不等;基因重叠区7处23bp,重叠碱基数在1～7bp之间。13个蛋白编码基因中,除COI起始密码子为GTG外,其余均以ATG为起始密码子;有8个基因(ND1、ND2、COI、ATP6、ATP8、ND4L、ND5、ND6)3'端有完全的TAA或TAG终止密码子,其它5个基因终止密码子为不完整的TA(ND3和ND4)或T(COⅡ,COⅢ,Cyt b)。除tRNASer(AGY)外,其余21个tRNA基因的二级结构均为典型的三叶草结构。预测的lrRNA二级结构共有6个结构域,53个茎环结构,srRNA二级结构包含43个茎环结构。控制区(D-loop)存在3个结构区:终止序列区(TAS)、中央保守区(CSB-F、CSB-D)和保守序列区(CSB-1、CSB-2、CSB-3),其中TAS与DNA复制终止相关,出现茎环结构。

The complete nucleotide sequence of mitochondrial genome of Pseudorasbora parva was determined using long PCR and sub-PCR approaches with 2 pair of long and 30 pair of short primers which were designed on the basis of its partial mtDNA sequences and other related fish species. The result showed that the entire mitochondrial genome of P. parva is 16 600 bp long with A＋T content 58.9 %. Its contained 13 protein coding genes, 22 tRNAs, 2 rRNAs, 1 control region named D-loop. All the 37 genes are conserved in the position in that of bony fish. tRNAGln, tRNAAla, tRNAAsn, tRNACys, tRNATyr, tRNASer （the second one）, tRNAGlu, tRNAPro and ND6 genes were encoded on the light strand （L strand）, and the remainders are on the heavy strand （H strand）. Genes are closely assembled one after the other, leaving a total of 64 bp in 13 intergenic spacers, ranging in size from 1-32 bp. The extremities of some genes overlap with each other by a few nucleotides, of which 7 overlaps range in size from size 1-7 bp, giving a total 23 bp. All protein-coding genes begain with ATG as initiation codon except COI using GTG. Most of the protein-coding genes, including ND1, ND2, COI, ATP6, ATP8, ND4L, ND5, ND6 had TAA/TAG as termination codon, while the others had incomplete termination codons TA/T. Most tRNA genes could form typical secondary structures except tRNASer （AGY）. There are six domains and 53 helices in secondary structures of lrRNA, 43 helices in secondary structures of srRNA respectively. In addition, there is an control region with 931 bp in length containing three structure regions of terminate associated sequence-TAS, central conservative blocks F/D and conservative sequence blocks 1-3, and the hairpin-loop structure of TAS occurred in with the termination of replication of DNA.