【作者】 陈合格；

【导师】 张轩杰；

【作者基本信息】 湖南师范大学 ， 动物学， 2005， 硕士

【摘要】 本文利用线粒体DNA分子标记技术研究了中华鳖、砂鳖和山瑞鳖线粒体DNA部分基因的序列并进行了比较分析,在分子水平证实了砂鳖为鳖属一新种并获得了这三种鳖各自的分子鉴定标记,主要结论如下: (1) 对中华鳖、砂鳖线粒体DNA 12S rRNA基因片段进行了序列测定和PCR-RFLP分析。结果表明:中华鳖、砂鳖线粒体DNA 12S rRNA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,两序列间共有13处碱基不同,序列差异率为2.31%,而中华鳖与砂鳖各自个体间的平均序列差异率分别为0.53%和0.36%,种间差异显著;用内切酶Msp I酶切两种鳖的12S rRNA基因片段,在砂鳖中可得到大小为519bp和43bp两个片断,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。 (2) 对中华鳖、砂鳖和山瑞鳖线粒体DNA 12S rRNA、16S rRNA基因进行了序列测定、序列的比较分析和系统进化分析。对三种鳖线粒体DNA两个rRNA基因全序列核苷酸差异率的分析表明:中华鳖与砂鳖12S rRNA基因序列间的差异率为4.0%,中华鳖、山瑞鳖间的序列差异率为11.0%,砂鳖、山瑞鳖间的序列差异率为10.8%;中华鳖与砂鳖16S rRNA基因全序列的差异率为4.0%,中华鳖、山瑞鳖间的序列差异率为13.2%,砂鳖、山瑞鳖间的序列差异率为12.8%。基于三种鳖mtDNA 12S rRNA、16S rRNA基因全序列的系统进化分析结果均支持砂鳖为鳖属一新种。 (3) 对中华鳖、砂鳖和山瑞鳖线粒体DNA细胞色素b基因进行了序列测定、序列的比较分析、系统进化分析和PCR-RFLP分析。结果表明中华鳖、砂鳖与山瑞鳖线粒体DNA Cytb基因的序列全长相同,均为1140bp,其A、C、G、T含量相似。序列间的差异率分析表明:中华鳖与砂鳖此基因序列间的差异率为7.7%,中华鳖、山瑞鳖间的序列差异率为15.0%,砂鳖、山瑞鳖间的序列差异率为15.9%,种间差异显著,且中华鳖与砂鳖的差异率相对较小。PCR-RFLP分析表明:用内切酶Nde I可准确鉴别砂鳖,而用内切酶BamH I则可准确鉴别更多还原

【Abstract】 Some genes of mitochondrial DNA of Pelodiscus sinensis , Pelodiscus axenaria and Palea steindachneri were cloned and sequenced by using molecular technology of mitochondrial DNA. Based on the comparative analysis on their sequence data, we confirmed Pelodiscus axenaria is a new species in Pelodiscus from the molecular level and got their molecular identification markers in three species of soft-turtles. The main results were summarized as follows:(1) The primers for Pelodiscus sinensis and Pelodiscus axenaria were designed and 562 base pairs of mitochondrial DNA 12S rRNA gene were amplified and sequenced. The results showed that the length of their sequence is the same, the A、 T、 G、 C contents are similar. 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 Pelodiscus. Restriction endonuclease analysis based on sequence data of this DNA fragment revealed the presence of polymorphic sites for Msp I endonuclease, there is one site for Msp I endonuclease in Pelodiscus axenaria, but none in Pelodiscus sinensis. The restriction profiles obtained by agarose gel electrophoresis when amplicons were cut with Msp I enzyme allowed the unequivocal identification between Pelodiscus sinensis and Pelodiscus axenaria.(2) The primers for 12S rRNA and 16S rRNA genes of Pelodiscus sin ensis, Pelodiscus axenaria and Palea steindachneri were designed and the complete sequences of the two rRNA genes were amplified and sequenced. Based on their sequence data of 12S rRNA gene, we can know the nucleotide percentage divergence is 4. 0% between Pelodiscus sinensis and Pelodiscus axenaria, 11.0% between Pelodiscus sinensis and Palea steindachneri, 10.8% between Pelodiscus axenaria and Palea steindachneri, whereas the nucleotide percentage divergence is 4. 0% between Pelodiscus sinensis and Pelodiscus axenaria, 13. 2% between Pelodiscus sinensis and Palea steindachneri, 12.8% between Pelodiscus axenaria and Palea steindachneri when the sequence data of 16S rRNA gene is analysised. The phylogenetic relationship of 12S rRNA and 16S rRNA genes of four species of soft-turtles was respectively described. All of the molecular phylogenetic trees indicated that Pelodiscus axenaria is more closely related with Pelodiscus sinensis than the other two species of soft-turtles and supported Pelodiscus axenaria is a new species in Pelodiscus.( 3 ) The primers for Pelodiscus sinensis , Pelodiscus axenaria and Palea steindachneri were designed and 1140 base pairs of mitochondrial DNA cytochrome b gene were amplified and sequenced. The results showed that the length of their sequence is the same, A> C G^ T contents are similar. Based on their sequence data, we can know the nucleotide percentage divergence is 7. 7% between Pelodiscus sinensis and Pelodiscus axenaria, 15. 0% between Pelodiscus sinensis and Palea steindachneri, 15. 9% between Pelodiscus axenaria and Palea steindachneri, whereas the individual nucleotide percentage divergence is 2. 37% in Pelodiscus sinensis , 0. 88% in Pelodiscus axenaria and 0. 18% in Palea steindachneri. The nucleotide percentage更多还原