Transcriptomic Analysis And Characterisation Of Four Stress-resistant Genes From The Housefly (Musca Domestica)

To generate a substantial EST dataset of Musca domestica, massively parallel pyrosequencing on the Roche454-FLX platform were used. This general information of the transcriptome can establish a fundamental resource for further research on adaption, antioxidation and immunologic mechanism in the fly. Based on EST of M. domestica, seven different genes including1heat shock protein (MdHSP70-1),2metallothionein (MdMT1, MdMT2), superoxide dismutase (MdSOD1, MdSOD2, MdSOD3) and rhodanese (MdRhodlikel) were cloned and sequenced. Quantitative PCR (qPCR), RNAi, recombinant protein, cell transfection and western technologies were used to research these genes, the main results are as follows:1. A substantial EST dataset was generated through massively parallel pyrosequencing method, thus a total of249,555ESTs with an average read length of373bp were obtained. These reads were assembled into13,206contigs and20,556singletons. Using BlastX searches of the Swissprot and Nr databases, we were able to identify4,814contigs and8,166singletons as unique sequences. Subsequently, the annotated sequences were subjected to GO analysis and the search results showed the majority of the query sequences were assignable to certain gene ontology terms. In addition, functional classification and pathway assignment were performed by KEGG and2,164unique sequences were mapped into184KEGG pathways in total.2. Using qPCR, the transcriptional profile of the different genes under heat shock, cadmium and bacteria stressful conditions was investigated. Increased maximum expression level of MdMT2was observed in response to heat shock1h, other genes maximum expression level were after heat shock recovery1h. Maximum expression level MdMT1and MdMT2were observed in response to10mM Cd, with the exception that MdRhodlike1was irregular trend, other genes reached the maximum levels at30mM for24h. The expression of MdHSP70and MdRhodlikel could be significantly induced by Escherichia coli or Staphylococcus aureus stimulation for24h, other genes significantly induced at60h. These results showed the seven genes different expression levels in different environmental response.3. Housefly feed on bacteia expressing dsRNA targeting the genes were abrogated by RNAi. Our results showed that high mortality happened in the larvae when treated with dsRNA at heat shock, Cd stress and bacterial invasion, suggesting that genes were potentially involved in the stress and immune responses of housefly and perhaps contributed to the protective effects against cellular injury.4. The MdSOD2gene was cloned into the prokaryotic expression vector to obtain the fusion proteins rMdSOD2. Between them, the activity of rMdSOD2was founded by visual assay methods. The present results provided new insights into MdSOD2of M. domestica.5. In order to further study these genes’function, a mammal expression vector pEGFP-N1with a green fluorescent protein was constructed and transformed into insect cells by liposomes method. The result of expression showed that MdSOD2and MdRhodlikel were located in the cytoplasm.6. Herein, to study the expression of MdMT1, MdSOD2and MdRhodlikel genes, a baculovirus expression system was constructed in insect cell SF9. Specific bands on western blot results revealed that the expression products were successfully detected.