山地麻蜥Sox与Dmrt基因保守区的克隆研究及其睾丸组织cDNA文库的初步构建-动物学专业论文.docxVIP

Studies on the Sox and Dmrt genes and Initial Construction of cDNA Library of Testis in Eremias breuchleyi Abstract Sox and Dmrt gene families participate in the development process of animal. The Sox genes form a large family which is characterized by a highly conserved DNA-binding and share a high sequence similarity with the HMG box region of the human Y chromosomal gene, SRY. They play important roles in embryonic development and sex differentiation. The Dmrt genes constitute a new gene family related to sex-determination. They encoded transcription factors characterized by a conserved zinc finger-like DNA-binding motif, the DM domain. In order to study the Sox and Dmrt genes of Eremias breuchleyi, degenerate PCR was used to amplify the Sox gene HMG-boxes and the conserved DM domains of Dmrt genes respectively. A database search for the cloned sequence, EbSox2, EbSox3, EbSox4, EbSox11, EbSox12, EbSox14 and EbSox21, revealed 96%, 88%, 94%, 99%, 96%, 98%, 97% identity with the homologous SOX genes of the human. Then, five different DM sequences were identified and named as EbDmrt1a, EbDmrt1b, EbDmrt3, EbDmrt4 and EbDmrt5 according to its name Eremias breuchleyi. The results indicated that Dmrt genes were highly conservative in phylogeny and had several members. To get full-length cDNA sequences of these genes for the study for sex-determining mechanism, a cDNA Library of Testis in Eremias breuchleyi was constructed initially with SMART technique. Agrose gel electrophoresis showed that the length of full-length cDNAs in this library was pooled mainly between 500~2000 base pairs; Using T/A cloning and choosing positive clones randomly, the double strands cDNAs were sequenced. The length of one sequence was 592bp which contains ORF of 162bp, 5’ end of non-translational region of 158bp and 3’ end of non-translational region of 272bp. This verified that this initial library was appropriate for constructing a cDNA library with high quality and can be used to screen and