中华鳖与砂鳖线粒体DNA 12S rRNA基因序列的比较分析和分子鉴定标记  

Comparative analysis of mitochondrial DNA 12S rRNA region between Pelodiscus sinensis and Pelodiscus axenaria and their molecular marker for identification

作　　者：陈合格[1] 刘文彬[1] 张轩杰[1] 

机构地区：[1]湖南师范大学生命科学学院,湖南长沙410081

出　　处：《水产学报》2005年第3期318-322,共5页Journal of Fisheries of China

基　　金：农业部"中华鳖标准"项目资助(1999-Q-1603161)

摘　　要：对中华鳖和砂鳖线粒体DNA12SrRNA基因进行了引物设计、PCR扩增、序列测定和PCRRFLP分析。研究结果表明:中华鳖、砂鳖线粒体DNA12SrRNA基因片段的碱基序列长度相同,均为562bp,其A、T、C、G含量相似,分别为209个(37.2%)、121个(21.5%)、145个(25.8%)、87个(15.5%)和207个(36.8%)、120个(21.4%)、145个(25.8%)、90个(16%)。两序列间共有13处碱基不同,序列差异率为2.31%,中华鳖与砂鳖各自个体间的平均核苷酸序列差异率分别为0.53%和0.36%,种间差异显著;用内切酶MspI酶切两种鳖的12SrRNA基因片段,在砂鳖中可得到大小为519bp和43bp两个片段,而中华鳖无此酶切位点,这可作为准确鉴别中华鳖和砂鳖的分子鉴定标记。从两种鳖线粒体DNA12SrRNA基因片段碱基的显著差异和MspI酶切位点的变化,可以进一步证明砂鳖是不同于中华鳖的鳖属一新种。The primers for Pelodiscus sinensis and Pelodiscus axenaria were designed by using the sequences of mitochondrial DNA 12S rRNA region of Dogania subplana, Chrysemys picta and Chelonia mydas, 562 base pairs of mitochondrial DNA were amplified and sequenced. The results show that the length of their sequence is the same, the A,T,G,C contents are similar,the number of A,T,G,C is 209(37.2%),121(21.5%),145(25.8%),87(15.5%)in Pelodiscus sinensis and 207 (36.8%),120(21.4%),145(25.8%),90(16%)in Pelodiscus axenaria. Based on their sequence data, we found there are 13 different nucleotide sites (percentage divergence is 2.31%) between Pelodiscus sinensis and Pelodiscus axenaria by using the software of ClustalW, whereas the average number of different nucleotide sites is 3 (percentage divergence is 0.53%) in Pelodiscus sinensis and 2 (percentage divergence is 0.36%) in Pelodiscus axenaria. The nucleotide percentage divergence is obvious between species, so it suggests Pelodiscus axenaria is a new species in Trionyx. Restriction endonuclease analysis based on sequence data of this DNA fragment revealed the presence of polymorphic sites for MspI endonuclease, there is one site for MspI endonuclease in Pelodiscus axenaria, but none in Pelodiscus sinensis. The restriction profiles obtained by agarose gel electrophoresis when amplicons were cut with MspI enzyme allowed the unequivocal identification of Pelodiscus sinensis and Pelodiscus axenaria.

关 键 词：中华鳖 砂鳖 12S RRNA基因 PCR-RFLP分析 分子鉴定标记 

分 类 号：S917[农业科学—水产科学]