Identification And Immune Response Analysis Of Rab11from Musca Domestica

Rabll is a small GTPase implicated in membrane traffic, immunolocalizes to the trans-Golgi network, cytoplasmic vesicles, and the base of rhabdomeres and plays an important rool in the budding, trasport, tethering, docking and fusion of the vesicles. Being a pest that carrys many pathogenic organisms, Musca domestica L.(Diptera:Muscidae) lives in and has adapts well to the conditions that full of pathogenic microorganisms. As the houseflies are ubiquitous and harmful to our health, research on the housfly and especially its unique immune system has been considered a new guideline to reveal the insect physiology and immunology, and the development of the pesticides.This thesis contains6parts.Part1:A new Rabll-like GTPase were cloned from the housefly based on dbEST and transcriptome sequencing. The deduced peptides were analyzed. The full-length cDNA of Rabll is1220bp, which contains a645bp complete open reading frame (ORF) and encodes a deduced214amino acid peptide. The SignalP result shows that the theoretical molecular weight of this peptide is24.29kD and the theoretical isoelectric point is5.52. The result of sequence alignments analysis showed that this gene shares97%identity with Drosophila melanogaste.Part2:A new-actin were cloned from the housefly by using RT-PCR and RACE methods. The deduced peptides were analyzed. The full-length cDNA of-actinis1380bp, which contains a1056bp complete open reading frame (ORF) and encodes a deduced352amino acid peptide. The SignalP result shows that the-actinhasn’t any signal peptides and the theoretical molecular weight of this peptide is39.53kD, moreover, the theoretical isoelectric point is5.47. The result of sequence alignments analysis showed that this gene shares more than94%identity with many other species. The expressions of-actin in different recovering time after heat shock were expressed similarly and not significantly different with that of the unstimulated control, suggesting that-actincan be use d as internal control in gene mRNA expression level.Part3:Using-actinand GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as the internal control, relative expressions of housefly Rabll gene in different tissues and different time after challenging with Staphylococcus aureus and Escherichia coli were measured respectively by RT-qPCR analyses. The expression data indicated that the Rabll mRNA expression were similar between the Escherichia coli group and the Staphylococcus aureus group, both up-regulated and reached the maximum in48h. Meanwhile, Rabll is highly expressed in hemocyte and the gut, which suggests that Rabll is closely associated with the housefly immune response.Part4:To reveal the functions of Rabll further, the Rabll prokaryotic expression vector was constructed and then induced and expressed in DE3. The Rabll was purified from the crude protein and then injected into the rabbit to make antibody.Part5:The housefly larve were adequately fed with HT115, which can express specifically dsDNA to knock down the expression of the Rabll, then challenged with Staphylococcus aureus and Escherichia coli respectively. The survival rate of the feed group is significantly reduced comparing to the unfeed group.Part6:The Rabll fluorescence expression vector was constructed and transfected into sf-9cells. Strong fluorescence was abserved in the fluorescence microscope, which suggests a fact that the Rabll is located in the structure of the membrane ubiquitously. This result parallelized the similar research about Rabll in other species and lays foundations for it’s further research in cells.