Cjoning And Prcfiminarv Study On The Function Relationships Of Genes Related To The Rac1/PI3K/Akt Pathway In Chinese Shrimp Fenneropenaeus Chinensis

The frequent outbreaks of aquacultural diseases severely hamper the development ofshrimp culture industry, resulting in tremendous economic losses. Intensive studies onshrimp immune mechanisms can provide a theoretical basis for designing effectivedisease-controlling strategies and thus are beneficial to the sustainable development ofshrimp culture industry. The Toll signal transduction pathway is of great importance indefending pathogens in invertebrates. The signal transduction factors Rac1, PI3K andAkt were discovered in the transcriptomic data of the Chinese shrimp Fenneropenaeuschinensis through bioinformatics analysis. These factors are related to theRac1/PI3K/Akt pathway, which is mediated by Toll receptors. Rac1, as a GTP-bindingprotein with a low molecular weight, is a critical component of numerous complexsignaling networks and plays important roles in a variety of cellular processes, includingcell migration, cell proliferation, cytoskeletal rearrangement and immune defense. PI3Kand Akt, as the downstream factors of Rac1, are important intracellular signaltransduction proteins, engaging in various cellular responses. In this thesis, the cDNAsequences of Rac1, PI3K catalytic subunit and Akt were obtained from the Chineseshrimp F. chinensis and the sequence characteristics of their deduced amino acids wereanalyzed. Their tissue expression patterns and transcription profiles in response todifferent pathogen challenges were analyzed. Also, their functional relations werestudied by using RNAi technique and the results suggested a vital role ofRac1/PI3K/Akt pathway in shrimp immunity. The progress obtained in this thesis arelisted as follows.The open reading frame (ORF) of Rac1gene in F. chinensis (FcRac1) is579bp,encoding192amino acids. It contains a RHO domain. Its deduced protein sequence demonstrated high conservation and phylogenetic analysis showed that it was closelyrelated to Rac1proteins in other arthropods. FcRac1had the highest transcription levelin the hemocytes and it could be detected in all types of hameocytes by in situhybridization. The transcription level of FcRac1was significantly up-regulated at1hour post Micrococcus lysodeikticus or Vibrio anguillarum challenge. During the courseof WSSV infection, the transcription profile of FcRac1was also changed. The presentdata indicated that FcRac1might play important roles in shrimp immune defenseagainst bacteria and virus.The ORF of PI3K catalytic subunit in F. chinensis (FcPI3Kc) is3222bp, encoding1073amino acids. Phylogenetic analysis showed that FcPI3Kc, which contains fivecharacteristic domains, is highly conserved. It had the highest expression level inshrimp hemocytes. The transcription level of FcPI3Kc was significantly up-regulated at1hour post Micrococcus. lysodeikticus or Vibrio anguillarum challenge and wasdown-regulated at3hours post WSSV challenge.The ORF of Akt in F. chinensis (FcAkt) is1536bp, encoding511amino acids. FcAktprotein contains a PH domain, a serine/threonine protein kinases catalytic domain and aser/thr-type protein kinases domain. FcAkt has a close phylogenetic relationship withAkt/PKB proteins in other arthropods. The transcription of FcAkt showed remarkablechanges after bacterial or WSSV challenge, suggesting that FcAkt might play importantroles in the innate immunity of shrimp.The transcription profiles of FcRac1, FcPI3K and FcAkt demonstrated high similarityafter M. lysodeikticus, V. anguillarum or WSSV challenge indicating that all of themthey may be closely related. After FcRac1was silenced by specific dsRNA injection,the transcription of FcPI3Kc was also significantly suppressed whereas the transcriptionof FcAkt was significantly up-regulated. The data further suggested that a certainconnection may exist among FcRac1, FcPI3Kc and FcAkt.