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陈合格, 刘文彬, 李建中, 张轩杰. 三种鳖线粒体DNA细胞色素b基因序列的比较分析[J]. 水生生物学报, 2006, 30(4): 380-385.
引用本文: 陈合格, 刘文彬, 李建中, 张轩杰. 三种鳖线粒体DNA细胞色素b基因序列的比较分析[J]. 水生生物学报, 2006, 30(4): 380-385.
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CHEN He-Ge, LIU Wen-Bin, LI Jian-Zhong, ZHANG Xuan-Jie. COMPARATIVE ANALYSIS OF MITOCHONDRIAL DNA CYTB GENE AND THEIR MOLECULAR IDENTIFICATION MARKERS IN THREE SPECIES OF SOFT2TURTLES[J]. ACTA HYDROBIOLOGICA SINICA, 2006, 30(4): 380-385.
Citation: CHEN He-Ge, LIU Wen-Bin, LI Jian-Zhong, ZHANG Xuan-Jie. COMPARATIVE ANALYSIS OF MITOCHONDRIAL DNA CYTB GENE AND THEIR MOLECULAR IDENTIFICATION MARKERS IN THREE SPECIES OF SOFT2TURTLES[J]. ACTA HYDROBIOLOGICA SINICA, 2006, 30(4): 380-385.
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三种鳖线粒体DNA细胞色素b基因序列的比较分析

COMPARATIVE ANALYSIS OF MITOCHONDRIAL DNA CYTB GENE AND THEIR MOLECULAR IDENTIFICATION MARKERS IN THREE SPECIES OF SOFT2TURTLES

    摘要
  • 摘要: 对中华鳖、砂鳖和山瑞鳖线粒体DNA细胞色素b基因进行了引物设计、PCR扩增、序列测定和PCR-PFLP(Polymerase chain reaction-restriction fragment length polymorphism)分析。研究结果表明中华鳖、砂鳖与山瑞鳖线粒体DNACytb基因的序列全长相同,均为1140bp,其A、C、G、T含量相似,分别为378个(33.2%)、322个(28.2%)1、22个(10.7%)、318个 (27.9%)、373个(32.7%)、324个(28.4%)、124个(10.9%)、319个(28.0%)、380个(33.3%)3、30个(28.9%)、127个(11.1%)、303个(26.7%)。同源性及序列差异率分析表明:中华鳖与砂鳖b基因序列的同源性为92.3%,中华鳖、山瑞鳖的为85.0%,砂鳖、山瑞鳖的为84.1%;中华鳖与砂鳖b基因核苷酸序列间的差异率为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%,而中华鳖、砂鳖、山瑞鳖各自个体间的序列差异率分别为2.37%、0.88%和0.18%,种间差异显著。用内切酶酶切分析其扩增产物,结果表明:用内切酶NdeⅠ可准确鉴别砂鳖,而用内切酶BamHⅠ则可准确鉴别山瑞鳖。内切酶NdeⅠ和BamHⅠ的联用分析,可使这三种鳖在分子水平都得到明确的鉴定。从三种鳖线粒体DNACytb基因核苷酸序列的显著差异和酶切位点的变化,可以进一步证明砂鳖是不同于中华鳖的鳖属一新种。

     

    Abstract: The primers for Pelodiscus sinensis, Pelodiscus axenaria and Palea steindachneri were designed by using the sequences of mitochondrial DNA tRNA2Glu, tRNA2Thr gene of Dogania subplana, Chrysemys picta and Chelonia mydas, 1140 base pairs of mitochondrial DNA cytb gene were amplified and sequenced.The results show that the length of their sequences is the same, A, C, G, T contents are similar, the number of A, C, G, T is 378 (33.2%),322 (28.2%),122 (10.7%),318 (27.9%-in Pelodiscus sinensis: 373(32.7%),324(28.4%),124(10.9%),319 (28.0%-in Pelodiscus axenaria and 380 (33.3%),330 (28.9%),127(11.1%),303(26.7%-in Palea steindachneri.Based on their sequence data, analysis with the software of Jellyfish, we can know: the nucleotide percentage homology is 92.3% between Pelodiscus sinensis and Pelodiscus axenaria, 85.0% between Pelodiscus sinensis and Palea steindachneri, 84.1% between Pelodiscus axenaria and Palea steindachneri; 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 nu-cleotide percentage divergence is 2.37% in Pelodiscus sinensis, 0.88% in Pelodiscus axenaria and 0.18% in Palea stein2 dachneri.Restriction endonuclease analysis based on sequence data of their DNAfragments revealed the presence of polymorphic sites for Nde Ⅰand BamH Ⅰendonucleases.The restriction profiles obtained by agarose gel electrophoresis when amplicons were respectively cut with Nde Ⅰand BamH Ⅰenzymes allowed the unequivocal molecular identification of Pelodiscus axenaria, Palea steindachneri and Pelodiscus sinensis.Based on their obvious nucleotide percentage divergence and the changes of poly2 morphic sites for Nde Ⅰand BamH Ⅰendonucleases in their sequences, it from the molecular level suggests Pelodiscus axe2 naria is a new species in Pelodiscus.

     

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