钦州湾牡蛎线粒体16S rRNA和COⅠ基因片段的序列变异分析

Sequence Analysis on Mitochondrial 16S rRNA Gene and CO Ⅰ Gene of Populations of Ostreidae from Qinzhou Gulf

利用线粒体16S rRNA基因和线粒体细胞色素氧化酶亚基Ⅰ(cytochrome oxidaseⅠ,COⅠ)基因片段初步研究钦州湾牡蛎(Ostrea)的物种组成。以特异引物进行PCR扩增,对扩增产物进行纯化、测序,分析表明,16S rRNA基因部分长度为413bp,COⅠ基因部分长度为535bp,2种牡蛎序列的碱基组成均显示出较高的A+T比例:16S rRNA基因59.8%;COⅠ基因60.5%。对位排序比较表明,16S rRNA片段种内个体间变异较小,存在7个变异位点,4种单倍型,其中包括5个转换位点突变,2个颠换位点突变;COⅠ片段有30个碱基存在变异,7种单倍型,其中包括16个转换位点突变,14个颠换位点突变。运用MEGA4软件计算出不同个体间的遗传距离,并构建了NJ和UPGMA系统树。香港牡蛎(Crassostrea hongkongensis)16S rRNA和COⅠ序列与白肉牡蛎的遗传距离均为0.000,有明巨牡蛎(Crassostrea ariakensis)16S rRNA和COⅠ序列和红肉牡蛎(red meat ostrea)的遗传距离分别为0.000和0.011,白肉牡蛎16S rRNA和COⅠ序列和红肉牡蛎之间的遗传距离分别为0.035和0.146。结果表明,钦州湾牡蛎分为2个不同的种,线粒体16S rRNA和COⅠ基因在种间存在明显的多态性,证实了16S rRNA和COⅠ基因序列适用于牡蛎的系统学分析。

Species composition of Ostrea from Qinzhou Gulf was studied based on mitochondrial 16S rRNA and mitochondrial cytochrome oxidase subunit Ⅰ gene （CO Ⅰ ） partial fragment. PCR amplified sequence with special primers. After the PCR outcome was purified, we determined and analyzed nucleotide sequence of 16S rRNA gene and CO Ⅰ gene, which was 413 bp and 535 bp, respectively. A high percentage of A＋T base composition of the two sequences was observed, which 16S rRNA gene was 59.8%, CO Ⅰ gene was 60.5%. Alignment indicated that variation level of intra-species was low in both sequences. 7 variable sites were detected in 16S rRNA gene fragment. There are 4 haloptypes, of which there were 5 transition sites and 2 transversion sites. 30 variable sites were detected in CO Ⅰ gene fragment, and there were 7 haplotypes, of which there were 16 transition sites and 14 transversion sites. The pair wise genetic distances were computed by MEGA4, NJ and UPGMA phylogenetic trees were obtained. Genetic distances between 16S rRNA gene, CO Ⅰ gene of C. hongkongsis and white meat oysters were 0.000 respectively. Genetic distances between 16S rRNA gene, CO Ⅰ gene of C. ariakensis and red meat oysters were 0.000 and 0.011 respectively. Genetic distances between 16S rRNA gene and CO Ⅰ gene of white meat oysters and red meat oysters were 0.035 and 0.146 respectively. The results indicated that Qinzhou Gulf population showed 2 different species, mitochondrial 16S rRNA and CO Ⅰ, showing obvious polymorphism, which are useful to systematic analyzing of Ostreidae.