Primary Study On Cloning And Subcellular Localization Of One Kind Of Mammalian Gonad Specific Expression Gene

This thesis studies issues related to a kind of mammalian gonad specific expression gene—GSE gene (Gonad-Specific Expression Gene) and it's subcellular localization in testis. It contains two parts: partⅠis review the literatures; partⅡincludes chapterⅡand chapterⅢ, which are experimental works.The objective of this study in chapterⅡwas to clone and analyze the GuangXi native buffalo GSE gene and its 3' cDNA end, which lay the foundations for further studies on the functions of buffalo GSE gene. A pair of primers for amplifying the conserved fragment of the buffalo GSE gene was designed according to the mouse GSE gene and other species similar genes from GeneBank. Then the total RNA which was extracted from adult buffalo testis was used as template to amplify the conserved fragment of the buffalo GSE gene by RT-PCR. After RT-PCR amplification, the amplified fragment (396 bp) was purified and inserted into the pMD18-T vector and sequenced. The results of Then an upstream primer was designed according to the 396 bp sequence, and the 3' cDNA end of buffalo GSE gene was amplified by 3' RACE with the upstream primer above. The results showed that the buffalo GSE gene obtained a 42-bp 3'UTR sequence. At last, a 700 bp sequence was obtained by piecing this fragment with the fragment above together. This was the longest fragment of buffalo GSE gene by now. Analysis of the deduced amino acid sequence of buffalo GSE protein by using Scansite software showed that polypeptide chain encoded by buffalo GSE gene contained a Shc protein structure of which having a SH2 domain (VKLSPGQYELLPPPV) and a PKC-μstructure (PPPVPKQASRSFVFRSTVQRFP), indicating that the GSE protein might have biological activities on phosphorylation and dephosphorylation.The objective of chapterⅢwas to detect the expression site of buffalo GSE gene in adult buffalo testis by tissue in situ hybridization, which in order to understand the functions of GSE gene during gametogenesis. The DIG-labeled cDNA probe was synthesized by using the buffalo GSE gene as template. After the quality detection of labeling efficiency and dot blotting, the probe could be used for in situ hybridization. At last, there were signal in basal compartment of contorted seminiferous tubules, no signal in adluminal compartment and in lumen after the hybridization of the probe with adult buffalo testis paraffin section. Comparing with cell types of contorted seminiferous tubules, it could be found that the GSE gene had expressed abundantly in primary spermatocytes, in which these germ cells were in meiosis. Therefore, the characteristic distribution and expression of GSE in these germ cells suggested that the GSE gene involved in the meiosis process.