汉寿中华鳖线粒体细胞色素b基因克隆及其遗传多样性分析

Cloning and Genetic Diversity Analysis of Mitochondrial Cytochrome b in Hanshou Trionyx sinensis

本文以汉寿中华鳖为研究材料,比较分析中华鳖线粒体细胞色素b基因(Cytb)与瑞鳖、砂鳖等的差异,为探讨其进化与遗传分子标记奠定实验基础。采用PCR技术,克隆汉寿中华鳖Cytb基因的序列,并检测其在不同组织以及不同生长发育时期的表达模式,最后通过分子进化树分析中华鳖与砂鳖的亲缘关系。通过克隆发现中华鳖细胞色素b基因的mRNA开放阅读框含有1 140 bp,其碱基组成为A+T的含量(61. 1%)高于G+C(38. 9%),编码379个氨基酸残基的序列。与山瑞鳖和砂鳖氨基酸序列比较发现,中华鳖与瑞鳖和砂鳖均存在一定差异; Cytb基因在中华鳖不同组织和各个不同发育时期中均有表达,且在精巢组织中表达量最高,在不同时期阶段呈"低-高-低"曲线表达模式;分子进化树分析发现汉寿中华鳖与砂鳖具有较强的亲缘关系,但是在Cytb中仍存在差异,所以能够很好的把它们区分开来。此外汉寿中华鳖与哺乳动物、鱼类和爬行类均存在巨大差异。结果表明,Cytb基因在探讨汉寿中华鳖的进化与遗传分子标记中具有重要意义,可作为分析中华鳖种属进化系统发育特性和群体遗传多样性的标记基因。

In this paper,Trionyx sinensis was used as research material,the difference of mitochondrial cytochrome b gene（Cytb） was compared and analyzed between Trionyx sinensis and Palea steindachneri or Pelodiscus axenaria. Cytb gene from HanShou Trionyx sinensis was cloned by using the PCR technology,and the expression patterns of different tissues and different growth stages were detected. Finally,the genetic relationship between Trionyx sinensis and Pelodiscus axenaria was used to analyze through molecular phylogenetic tree. It is found that the mRNA open reading frame of Cytb gene of Trionyx sinensis was found to contain 1 140 bp through cloning,encoding 379 amino acids. And the content of the bases for A ＋ T（61. 1%） was higher than that of G ＋ C（38. 9%）. The amino acid sequential comparison with Palea steindachneri or Pelodiscus axenaria indicated that there were obvious differences. Moreover,Cytb gene was expressed in different tissues and different developmental stages of Trionyx sinensis. Besides,the highest expression was found in testis tissue. Furthermore,its expression level exhibited a＂low-high-low＂pattern at different development stages. Further molecular evolutionary tree analysis suggested that Trionyx sinensis has a close relationship with Pelodiscus axenaria. However,there was distinguishable difference in Cytb between these two species. In addition,in view of the obvious difference in Cytb among mammals,fish and reptiles,Cytb might play on important role in the evolution and genetic molecular marking in trionyx sinensis.