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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。
The species composition of Ostrea in Qinzhou Bay was preliminary studied by using mitochondrial 16S rRNA gene and mitochondrial cytochrome oxidase Ⅰ (COⅠ) gene fragment. The specific primers were used for PCR amplification. The PCR products were purified and sequenced. The results showed that the partial length of 16S rRNA gene was 413 bp and the length of COⅠ gene was 535 bp. The base composition of two oyster sequences showed high A + T ratio: 59.8% of 16S rRNA gene; 60.5% of COI gene. The comparison of para-alignment showed that the 16S rRNA fragments within the species variation was small, there are seven mutation sites, four haplotypes, including five conversion site mutations, two transversion site mutations; COI fragments have There are 30 base variations, seven haplotypes, including 16 mutations in the conversion site, 14 transversion site mutations. Using MEGA4 software to calculate the genetic distance between different individuals, and constructed NJ and UPGMA phylogenetic tree. The genetic distances between 16S rRNA and COⅠ sequences of Crassostrea hongkongensis and white meat oysters were both 0.000. The genetic distances between 16S rRNA and COⅠ sequences of Crassostrea ariakensis and red meat ostrea were 0.000 and 0.011, respectively The genetic distance between 16S rRNA and COⅠ sequences of white meat oyster and red meat oyster was 0.035 and 0.146, respectively. The results showed that oysters in Qinzhou Bay were divided into two different species. There were obvious polymorphisms in mitochondrial 16S rRNA and COⅠ gene, which confirmed that 16S rRNA and COⅠ gene sequences were suitable for the systematic analysis of oyster.