【摘要】 利用RT-PCR和cDNA末端快速扩增法(RACE)分离、克隆奥利亚罗非鱼(Oreochromis aurea)睾丸DMT(DM-do-main gene in testis)基因,并进行序列测定与分析。结果表明,该基因cDNA序列全长1 260 bp,包括74 bp 5’非翻译区,879 bp阅读框以及含poly(A)信号AATAAA的307 bp 3’非翻译区,阅读框共编码292个氨基酸。序列同源性分析表明,不同进化地位动物的DMRT1基因DM域编码序列存在高度同源性,显示DMRT1基因在系统进化上高度保守。生物信息学分析表明:DMT基因编码蛋白无信号肽,无跨膜区域,有多个蛋白激酶C、酪蛋白激酶Ⅱ磷酸化位点和N-糖基化位点,推测其可能在细胞信号传导中发挥作用,且其生物活性可能接受信号途中多种信号的调控。该基因的成功克隆及生物信息学分析不仅为DMRT1基因的分子进化和相似性比较研究提供了新的材料,而且对进一步研究其编码蛋白的结构与功能以及其在鱼类性别调控中的作用具有重要意义。[中国水产科学,2007,14(1):23-31]更多还原

【Abstract】 DMRT gene constitutes a new gene family related to sex-determination.They were isolated by virtue of its homology to doublesex gene from Drosophila,Mab-3 from Caenorhabditis elegan and human DMRT1 gene.DMRT1 can encode putative transcription factors characterized by a conserved zinc finger-like DNA-binding motif,the DM domain.In this study,RT-PCR and RACE were used for the cloning of full length DMT(DM-domain gene in testis) cDNA from testis of Oreochromis aurea.Two pairs of primers P1,P3 and P2,P3 were designed according to the sequence of fish DM domain.A 120 bp conserved sequence of DMT cDNA was cloned.The other pair of primers P4 and P5 were designed according to the conserved sequence for 3’ RACE.And other three gene specific primers 1,2 and 3 were designed according to the sequence of 3’ end cDNA.Sequence analysis revealed a 1 260 bp full-length cDNA sequence containing 74 bp 5’-untranslated region,307 bp 3’-untranslated region and 879 bp open reading frame(ORF) and this sequence was encoded by 292 amino acids.The deduced amino acid sequence aligned with those of DMRT1 genes from different species showed high degree of sequence homology as phylogenic tree revealed.The amino acid sites of DM domains may form C2/H2 model zinc-finger structure to bind specific DNA sequences and regulate sex differentiation and development.It contained a male specific motif,which was well conserved among numerous DMRT1 genes but absent in DMO,indicating that DMT represented a male-type DMdomain gene and played an important role in sex differentiation and sex development.The bioinformatics analysis revealed that the predicted protein had no signal peptide and notable transmembrane region.It contained much PKC-PHOSPHO-SITE,CK2-PHOSPHO-SITE and ASN-GLYCOSYLATION-SITE,implicating that they could play some roles during cellular signal conduct and their activities might be related to the regulation of many signals during signal conducting route.The successful cloning of this gene not only provides new material for researches on DMRT1 molecular evolution and similarity comparison of DMRT1 gene,but also is very important for further study of structure and function of DMT in Oreochromis aureus and the mechanism of sex control of fish.[Journal of Fishery Sciences of China,2007,14(1):23-31]更多还原