卵形鲳鲹线粒体COI基因全长序列的克隆与分析

Cloning and Sequence Analysis of Trachinotus ovatus Mitochondrial COI Gene

根据近源物种线粒体序列的同源比对,在COI基因上下游保守区域设计一对通用引物COF/COR。以卵形鲳鲹肌肉总DNA为模板,PCR扩增获得特异的DNA片段,经克隆、测序和比对证实该片段包含了卵形鲳鲹线粒体COI基因完整编码区1551bp。对5个个体分别测序后比对,发现卵形鲳鲹COI基因DNA序列在钦州湾种群个体间至少存在7个变异位点,使5个个体分别具有5种不同的单倍型。将卵形鲳鲹种内不同个体及鲹科其他物种的CO I核酸序列进行比较分析,根据比对结果构建鲳鲹科各物种的系统进化树,支持鲹科下设四个亚科(鲹亚科,鰤亚科,鲳鲹亚科,鰆鲹亚科)的分类系统。通过COI基因遗传距离计算发现,卵形鲳鲹种内不同地理种群个体间遗传距离为0.001～0.005,显著低于Hebert等所推荐的物种鉴定最小种间遗传距离2%,而鲹科不同物种间遗传距离大于0.044,与形态学分类一致,说明COI作为卵形鲳鲹DNA条形码应用是可行的。

Based on homology alignment of mitochondrial sequence from neighbor species, a pair of conserved primers were designed on the flanking region of COI gene. The PCR amplification product was cloned to PGEM-Teasy vector and sequenced. Blast on NCBI confirmed that the sequence contain whole 1551 bp ORF of COI gene. Alignment of sequence from 5 fishes of the same population revealed at least 7 variation sites , which formed 5 different genotypes. Based on the multiple alignment of COI sequences of Trachinotus ovatus and other Carangidae species, a phylogenetic tree was constructed which supported the idea that Carangidae could be classed into four subfamilies： Caranginae, Seriolinae, Trachinotinae and Choriueminae. Genetic distance among Trachinotus ovatus from different populations was 0.001 -0.005, significantly lower than the distance between spices recommended by Hebert of 2 %. While the genetic distance among different Carangidae fishes spices were longer than 0.044, indicating that COI gene was a useful DNA harcoding for Trachinotus ovatus.