| Postgraduate:Jiang YuAdvisor:Liu WeiquanSome changes in transcriptional process of the early embryos proecssed by transgene were theoretically of most possibility occurred, and these changes such as the appearance of new genes would~ be relative to the fate of targeting gene. More exactly, they would affect the integration or degradation of targeting gene.In this paper, the transcriptional process of the early housefly embryos processed by transgene was studied and compared with the non-processed embryos by Differential Display(DD). Firstly, the reaction paramters were optimized and the silver staining DD was founded. In this part, the best working concentrations of template, MgCI2 and dNTPs were determined by the idea] results of sequencing gel after silver staining. Secondly, the housefly embryos were electroporated with eukaryotic expression plasmids梡cmGSl containing GFP (Green Fluorescent Protein) and ABP (Antibacterial Peptides) fusion gene. The situation of gene expression was studied by DD before differentiation of the embryos, and the differences between the normal and processed group were found. Three differential fragments of transgenic groups were cloned. The results of sequencing and homology analysis showed that one of them was a part of cytochrome C gene of Musca do,nestica, and the others 240 nt and 432 nt were new sequences. Thirdly. significant signals were observed when the two new fragments (genes) were hybridized with housefly genome by dot-blot. The results indicated that they were the part of housefly genome. Their sequences had been signed in the Genbank and their registered codons are AY030235 and AY030236, respectively. Finally, the fragment of cytoehrome C gene was confirmed by Northern blot. The result showed that the gene was expressed in both testing and normal embryos, which would be a quantity difference: the cDNA quantity of cytochrome C expressed in testing group was more than in normal group.
|
PDF Full Text Request