| Summary: | 博士 === 國立中興大學 === 昆蟲學系 === 89 === The male accessory gland (MAG) protein that modulates the host-seeking behavior and stimulating oviposition of female Aedes aegypti (L.) mosquitoes were found to be a peptide of 31.7 kDa. This peptide also prevented subsequent mating behavior and weakly stimulated oviposition. For constructing cDNA library of male accessory gland of Ae. aegypti, RNA was isolated from adult male 3 days after feeding on sucrose, using the guanidinium thiocyanate extraction. Double stranded cDNA was synthesized using total RNA from 1,000 males as the template and directly cloned into the pBluescript SK(+) phagemid and packaged using the Gigapack III Gold Packaging Extract. Approximately 200,000 plaques were obtained and used to infect E. coli strain MRF’ in order to amplify the library. The library was plated using E. coli strain XL1-Blue and screened with the mosquito 31.7 kDa (Yeh317) polyclonal antibody, and goat anti-rabbit IgG conjugated with alkaline phosphatase. Screening of the cDNA library is still undertaking at present.
The Yeh317 protein was purified and characterized from the mosquito, Ae. Aegypti, by SDS-PAGE and Coomassie brilliant blue staining. The specificity of the polyclonal antibodies raised in rabbits to the purified Yeh317 protein was verified by immunoblot analysis of crude male accessory gland extracts and the protein was separated by SDS-PAGE. The N-terminal amino acid sequence of the purified mosquito male accessory gland 31.7 kDa protein (15 amino acids) had 79% identity to the derived amino terminal sequence of the CG6041 gene product of Drosophila melanogaster and 47% identity to the derived amino terminal sequence of 3al cDNA of midgut of Ae. aegypti that codes for a putative trypsinogen. The protein is a soluble protein and detected only in the supernatant of tissue homogenate. For in situ localization of the protein, laser-scanning microscope (Confocal) was used to observe cross-sections of MAG. This protein was found as distributed in the whole accessory gland. Subsequent work labeled males with goat anti-rabbit conjugated FITC to the protein and, allowed these males to copulate with virgin females. Detection of fluorescence reaction in female spermathecae demonstrates that the protein transferred during copulation.
The crude MAG protein plays like a protease ability to digest GST protein. MAG protein could be digested with Yeh317 protein, therefore, use biochemistry method of in vitro study to prove that the protein from male accessory gland of Ae. aegypti which could destroy another MAG protein of Ae. aegypti, that demonstrate the protein could play like a protease activity to digest denature MAG protein, denature Yeh317 protein and Drosophila GST protein. Under room temperature condition of 27℃, the protein could digest GST protein and denatured Yeh317 protein. It might infer that in nature condition the female mosquito receptivity to further mating and, removing or destroying sperm of previous mates.
The MAG Yeh317 protein landmarks have been developed so that this protein is transferred during mating. It was also demonstrated that Yeh317 protein could transfer during venereal transmission into spermathecae of gravid mosquitoes and carry over to her offspring, which unambiguously identified and oriented by fluorescence in situ hybridization (FISH) and digital imaging microscopy. In this study, it is indicated that the Yeh317 protein could be carried over to female mosquito during copulation, and the protein was found in embryo and ovaries. The protein even could be found in head part of lateral retractor of flabellum, median phageal ganglion, deutrocerebrum and protocerebrum of the hatched first instar larva.
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