挂榜山小鲵线粒体基因全长测定及小鲵科系统发育分析
摘要
线粒体是存在于绝大多数真核生物细胞内的一种基本的、重要的细胞器。脊椎动物线粒体DNA (mitochondrial DNA, mtDNA)是共价闭合的环状双链DNA,能进行自我复制,具有严格的母系遗传特性,在世代传递过程中不发生基因重组。mtDNA已被广泛应用于脊椎动物进化遗传学和种群遗传学的研究,为从分子水平上解决物种的系统发育问题提供了理想的研究材料。
挂榜山小鲵(Hynobius guabangshanensis)隶属于两栖纲(Amphibia)、有尾目(Urodela)、小鲵科(Hynobiidae)。本研究通过通用引物测出几个基因片段然后通过LA-PCR扩增出两个基因之间的长片段,再用PCR引物步移测序等方法,测定了挂榜山小鲵线粒体全长。测序结果利用DNAStar软件包中的Megalign程序对同源序列进行排列,并经人工仔细核查。实验分析结果表明：挂榜山小鲵线粒体全长为16408bp,其中A为5484个,占33.4%；C为3478个,占21.2%；G为2255个,占13.7%；T为5191个,占31.6%。A+T含量明显大于G+C含量。
小鲵科物种截止2009年10月共有19个物种测定了线粒体基因全序列。本文利用MEGA4.0对NCBI上19个小鲵科物种线粒体基因全序列进行了比对,并选取了线粒体上几个基因进行系统进化分析,计算不同序列间的碱基组成、变异位点、简约信息位点数、转换／颠换比等等；用MEGA4.0软件基于P-distance模型构建19个小鲵科物种的遗传距离,采用Kimura2-parameter模型最大简约法(Maximum-Par simony)和邻接法(Neighbor-Joining Method)分别构建MP分子系统树和NJ分子系统树。
Mitochondrial is an essential organelle existed in most of eucaryote cells.Vertebrate mtDNA is a covalently closed circular double strand DNA,which can self-duplicate in a strict maternal inheritance way and does not recombinate in multigenerational transmission process.MtDNA has extensive used in vertebrate population genetics and evolutionary genetics,which provides a perfect research materials for resolving phylogeny on molecular level.
Hynobius guabangshanensis belongs to Hynobiidae,Urodela, Amphibia. This study has sequenced the complete mitochondrial genomes of H. guabangshanensis. First of all,several gene segments were sequenced with general Primer,then the complete mitochondrial genomes were sequenced and analyzed with the Long-PCR and Primer Walking in this research. Homologous sequence was arrangeed with MegAlign——a procedure of DNAStarlne and then with carefully Manual check to obtain complete mitochondrial genome.The result of complete mitochondrial genome of Hynobius guabangshanensis indicate:The length of H. guabangshanensis mtDNA is 16 408bp, among which A is 5484, account for 33.4%;C is 3478, account for 21.2%;G is 2255,account for 13.7%;T is 5191,account for 31.6%. The content of
A+T is obviously more than G+C.
Until October 2009,there are 19 complete mitochondrial genomes of Hynobiidae have sequenced. These 19 mtDNA of Hynobiidaes were analyzed and compared：including variable site, parsimony informative site, pairwise sequence alignment Pairwise P-distance.The molecular phylogenetic tree was constructed with MEGA4.0.The genetic diversity parameters of CO I,Cyt b and 12S+16S rRNA in 19 Hynobiidae species were anayzed including variable site, conserved site,Parsim-info site and Singleton site. Neighbor Joining tree and Maximum Parsimony tree(by transition and transivertion) were reconstructed using the Kimura 2-Parameter model in Mega 4.0,Numbers above branches indicate percentage bootstrap values for 1000 replicates (values>50%shown).
挂榜山小鲵(Hynobius guabangshanensis)隶属于两栖纲(Amphibia)、有尾目(Urodela)、小鲵科(Hynobiidae)。本研究通过通用引物测出几个基因片段然后通过LA-PCR扩增出两个基因之间的长片段,再用PCR引物步移测序等方法,测定了挂榜山小鲵线粒体全长。测序结果利用DNAStar软件包中的Megalign程序对同源序列进行排列,并经人工仔细核查。实验分析结果表明：挂榜山小鲵线粒体全长为16408bp,其中A为5484个,占33.4%；C为3478个,占21.2%；G为2255个,占13.7%；T为5191个,占31.6%。A+T含量明显大于G+C含量。
小鲵科物种截止2009年10月共有19个物种测定了线粒体基因全序列。本文利用MEGA4.0对NCBI上19个小鲵科物种线粒体基因全序列进行了比对,并选取了线粒体上几个基因进行系统进化分析,计算不同序列间的碱基组成、变异位点、简约信息位点数、转换／颠换比等等；用MEGA4.0软件基于P-distance模型构建19个小鲵科物种的遗传距离,采用Kimura2-parameter模型最大简约法(Maximum-Par simony)和邻接法(Neighbor-Joining Method)分别构建MP分子系统树和NJ分子系统树。
Mitochondrial is an essential organelle existed in most of eucaryote cells.Vertebrate mtDNA is a covalently closed circular double strand DNA,which can self-duplicate in a strict maternal inheritance way and does not recombinate in multigenerational transmission process.MtDNA has extensive used in vertebrate population genetics and evolutionary genetics,which provides a perfect research materials for resolving phylogeny on molecular level.
Hynobius guabangshanensis belongs to Hynobiidae,Urodela, Amphibia. This study has sequenced the complete mitochondrial genomes of H. guabangshanensis. First of all,several gene segments were sequenced with general Primer,then the complete mitochondrial genomes were sequenced and analyzed with the Long-PCR and Primer Walking in this research. Homologous sequence was arrangeed with MegAlign——a procedure of DNAStarlne and then with carefully Manual check to obtain complete mitochondrial genome.The result of complete mitochondrial genome of Hynobius guabangshanensis indicate:The length of H. guabangshanensis mtDNA is 16 408bp, among which A is 5484, account for 33.4%;C is 3478, account for 21.2%;G is 2255,account for 13.7%;T is 5191,account for 31.6%. The content of
A+T is obviously more than G+C.
Until October 2009,there are 19 complete mitochondrial genomes of Hynobiidae have sequenced. These 19 mtDNA of Hynobiidaes were analyzed and compared：including variable site, parsimony informative site, pairwise sequence alignment Pairwise P-distance.The molecular phylogenetic tree was constructed with MEGA4.0.The genetic diversity parameters of CO I,Cyt b and 12S+16S rRNA in 19 Hynobiidae species were anayzed including variable site, conserved site,Parsim-info site and Singleton site. Neighbor Joining tree and Maximum Parsimony tree(by transition and transivertion) were reconstructed using the Kimura 2-Parameter model in Mega 4.0,Numbers above branches indicate percentage bootstrap values for 1000 replicates (values>50%shown).
引文
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