中国大鲵Dmrt基因DM结构域的克隆及序列分析
CLONING AND SEQUENC ING DM DOMA IN OF THE DM RT GENE FROM THE CHINESE GIANT SALAMANDER, ANDRIAS DAV ID IANUS
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摘要: Dmrt基因家族是一个与性别决定相关的基因家族,该家族成员共有一个具有DNA结合能力的保守基序-DM结构域.为了进一步探讨该家族在系统进化中的保守性,本研究通过简并PCR技术,扩增并克隆了中国大鲵(An-drias davidianus)基因组中的DM结构域.序列分析显示,中国大鲵基因组中存在Dmrt基因的DM结构域.其核酸序列与猕猴、青鳉、人、小鼠、牛、热带爪蟾相应Dmrt基因DM结构域的相似性分别为91%、92%、92%、89%、91%、84%.其蛋白序列与上述物种的相似性均为91%,表现为4个氨基酸的变异,即第19、34、36和45位的精氨酸分别由半胱氨酸、谷胱酰胺、色氨酸和谷胱酰胺所取代.这些氨基酸的变化对其蛋白总体的三维构型没有显著影响.聚类分析结果表明,不同进化地位物种的Dmrt基因DM结构域编码序列存在高度的同源性,显示Dmrt基因在系统进化上的高度保守.Abstract: The Dmrt genes constitute a new gene family related to sex-determination, which encoded putative transcription factorswith a conserved DM domain that initiates genetic process in early development through itsDNA binding ability. So far, the Dmrt genes have been discovered in awide range of animal species, such as fish, amphibian, reptiles, birds, and mammals, which evidently revealed the evolutionary conservation of Dmrt gene family. Amphibians is a key in the evolu-tion during the aquatic and the terrestrial, the research of Dmrt gens in Andrias davidianus, which was a very important group in phylogenic evolution, has not been reported. In order to confirm the evolutionary conservation ofDmrt gen family in Andrias davidianus, degenerate PCR primers, designed from a highly conserved region of aligned multiple sequences, was successfully used for the amplification of the DM domain of the Dmrt gene from the Chinese giant salamander (Andrias davidianus). A DNA fragmentwith about 140 bp in length was obtained by PCR using A. davidianus genomic DNA as template. The DNA was cloned and its nucleotideswere determined by sequencing. Homology searching against databases confirmed its identity as Dmrt gene fragment from the A. davidianus. The sequence comparison analysis showed that the Andrias Dmrt shared high sequence similaritywith Dmrt genes from M acaca mulatta (91%), Oryzias latipes (92%), Ho-mo sapiens (92%), M us musculus (89%), Bos taurus (91%) and Xenopus tropicalis (84%), but its amino acid se-quence remained at a similar level with other Dmrts (91%). The Arginine of location 19, 34, 36 and 45 was replaced with cysteine, Glutamine, Tryptophane and Glutamine respectively. The results further indicated that Dmrt genes were highly conserved in phylogeny and the strong evolutionary conservation of this gene family suggested thatDmrt genesmight be importance in developmental process.
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Keywords:
- Andrias davidianus /
- RT2PCR /
- Dmrt /
- DM domain
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