Functional analysis of CAS1、ELMO and F-box/LRR in the yellow fever mosquito, Aedes aegypti

• Main Authors: De-Chang Lin, 林德昌
• Other Authors: 蕭信宏
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/78211997785079697783

碩士 === 國立臺灣大學 === 微生物學研究所 === 98 === It has been widely accepted that invertebrates contain only innate immunity but no adaptive immunity. The innate immunity includes phagocytosis, encapsulation, melanization and secretion of antimicrobial peptides (AMPs). There are five AMPs called Attacin, Cecropin, Defencin, Diptericin and Gambicin in the mosquito Aedes aegypti and these AMPs are regulated by Toll or Imd pathway. In mosquitoes, vitellogenesis and metamorphosis are important life cycle that can be researches decreasing vectore-borne diseases. Previous cDNA microarray analysis revealed that CAS1, ELMO and F-Box/LRR are simultaneously up-regulated with Inhibitor of Apoptosos 2 (IAP2), an important regulator of IMD pathway, upon bacteria challenge in Anopheles gambiae. CAS1 was associated with acetylation in hunan and fungus－Cryptococcus neoformans. It is also called acetyltransferase. ELMO was shown to be involved in D. melanogaster development and cytoskeleton stability. It was demonstrated to affect phagocytosis in mammals. Previous research showed that F-box may play important role in protein-protein interaction and it is an important factor in ubiquitylation. Therefore, we ought to explore the functions of CAS1, ELMO and F-box in the mosquito A. aegypti. First, we made use of RNA interference (RNAi) technique to silence the mRNA expression of ELMO and F-box in A. aegypti, followed by the challenge of Staphylococcus aureus or Escherichia coli. The survival assay was performed to analyze the mosquito resistance to bacteria. Our results revealed that Aedes aegypti showed resistance to S. aureus in the absence of ELMO and F-Box. Therefore, we speculated that ELMO and F-box may serve as negative regulators in Toll pathway. Next, the expression of Cecropin A, a downstream target of Toll pathway, was examined. The results showed that silencing of ELMO resulted in the over-expression of Cecropin A upon S. aureus challenge, suggesting that ELMO negatively regulateof the expression of Cecropin A. Then we made use of FITC-labeled bacteria to observe the effect of phagocytosis in A. aegypti. The result showed that silencing of ELMO can increase the phagocytic ability to S. aureus in the mosquito. It’s suggested that ELMO played a negative role in phagocytosis to Gram positive bacteria. In addition, we made use of RNAi technique to silence the mRNA expression of CAS1 and F-box in A. aegypti, followed by feeding blood meal. The result showed that silencing of CAS1 decreased vitellogenin expression and egg production. Finally we use RNAi technique to silence the ELMO and F-box in larvalstage of Aedes aegypti and calculate the ratio of mosquito emergence. The result revealed that silencing of F-box resulted in the decrease of metamorphosis rate in larval stage and metamorphosis genes (broad,E75b, JHA15) expression in adult mosquitoes.