蛤蜊科3种贝类16S rRNA基因片段及ITS2核苷酸序列分析

利用PCR技术分别扩增连云港及启东沿海蛤蜊科的西施舌(Coelomactra antiquata)、中国蛤蜊(Mactrachinensis)和四角蛤蜊(Mactra veneriformis)3种双壳贝的16S rRNA基因片段和ITS2核苷酸序列,测序后用DNA star软件分析了核苷酸差异。结果显示:三种贝类16S rRNA基因片段长度相同,均为306bp(去除引物),核苷酸存在多态性,共有45个变异位点,54个核苷酸发生了变异,全部为碱基置换。西施舌与中国蛤蜊此片段核苷酸的同源性为88.9%,与四角蛤蜊的同源性为88.6%,中国蛤蜊与四角蛤蜊的同源性为90.6%。三种蛤蜊ITS2序列分别为390 bp(西施舌)4、41 bp(四角蛤蜊)和466 bp(中国蛤蜊),存在长度多态性,ITS2核苷酸差异分析结果显示,西施舌与中国蛤蜊的同源性为70.9%-71.1%,西施舌与四角蛤蜊的为70.5%-71.0%,中国蛤蜊与四角蛤蜊的同源性为88.1%-88.8%。ITS2序列分析结果与16S rRNA基因片段分析结果一致,2种分子分析法均显示中国蛤蜊与四角蛤蜊的亲缘关系近。

Sequence Analysis on Mitochondrial 16S Rrna Gene Fragments and ITS2 Loci in Three Species of Mactradae (Mollusca: Bivalvia)

The PCR technique was used to amplify the mtDNA 16S rR NA gene fragments and ITS2 nucleotides in Coelomactra antiquata,Mactra chi nensis and Mactra venerifomis (Mollusca: Bivalvia) from the coast of Lia nyungang and Qidong.The PCR products were purified and sequenced.Alignments were manipulated using DNAStar software and refined manually where necessary.S equence analysis was performed using MEGA3.1 software.As a result,306 bp nucl eotide sequences of 16S rRNA gene fragment were obtained,45 varied sites in the fragments were detected.The percentage of the identity of the 16S rRNA gene fr agments is 88.9%,between C.antiquata and M.chinensis,88.6% between C.antiquata and M.veneriformis,and 90.6% between M.chinensis and M veneriformis.The length of ITS2 ranged from 390 to 466bp,obviously varied.The percentage of the identity of ITS2 nucleotides is 70.9%-71.1% bet ween C.antiquata and M.chinensis,70.5%-71.0% between C.antiquata and M.veneriformis,and 88.1%-88.8% between Mactra chinensis and M.veneriformis.The results suggested that it is closer between Mactra ch inensis and M.veneriformis based 16S rRNA gene fragments and ITS2 sequenc es among the three clams,It is also demonstrated through the genotype analysis t hat each species has its specific nucleotide sequence.The results suggest that the 16S rRNA gene fragment and ITS2 locus are the useful molecular markers in id entification of species,hybrids,larvae and tissue samples of the three clams.