| As one of the most valuable aquaculture species in China, grass carp(Ctenopharyngodon idella) exerting economical function as well as protein resources. However, as a subsequence of environmental pollution, the vulnerability to pathogen infection of which would cost us an enormous lost. From an aquacultural perspective, it is essential that the disease-resistance improved varieties to be selected for grass carp breeding. Among which, a crucial job is to investigate the function of potential immunerelated genes thus achieve a research purpose as molecular assisted selective breeding.Rho GTPase has been demonstrated to play fundamental roles in a vast array of cellular processes, such as cytoskeletal rearrangement and phagocytosis in mammals. However, no study has reported the immunological function of GTPase in grass carp. So, two GTPases from grass carp was identified and their immunological function had been analyzed in our dissertation.1. Rac1, a Rho GTPase, serves critical immunological functions in mammals. Here, a Rac1 homolog(gc Rac1) was identified in grass carp(Ctenopharyngodon idella). The fulllength 2023-base pair(bp) gc Rac1 c DNA contained a 579-bp open reading frame encoding a 192-residue protein, including a conserved RHO domain and nuclear localization signal. The gc Rac1 protein showed relatively high nuclear expression and phylogenetically clustered with Danio rerio Rac1. The gc Rac1 transcript showed wide tissue distribution and was inducible by Aeromonas hydrophila in vivo and in vitro; its expression also fluctuated with LPS or flagellin stimulation in vitro. With gc Rac1 over-expression, gc PAK1, gc IL1-β, gc TNF-α and gc IFN were up-regulated by A. hydrophila and bacterial PAMPs induction, while gc Rac1 knockdown decreased these transcripts after A. hydrophila challenge. Overexpression of gc Rac1 reduced, while its suppression facilitated, bacterial invasion. Moreover, gc Rac1 could activate NF-κB signaling. These findings implicate the vital role of gc Rac1 in grass carp innate immunity against pathogens.2. Cdc42, a crucial member of the Rho GTPase family, serves critical functions in a variety of cellular processes, including immunological responses to pathogens in mammals. To illustrate the roles of Cdc42 in piscine immunity, a homolog of the Cdc42 gene(Ci Cdc42), was identified in grass carp. The full-length Ci Cdc42 c DNA sequence obtained was 1955 base pairs(bp) with an open reading frame of 576 bp encoding a protein of 191 amino acids, which contains a conserved RHO domain and a nuclear localization signal. Meanwhile, the subcellular localization of Ci Cdc42 illustrated its relatively higher expression in the nucleus than other regions. In the phylogenetic analysis, Ci Cdc42 clustered closely with Cdc42 of Danio rerio and shared a high conservation between species. Expression of Ci Cdc42 was distributed widely in ten tissues tested and could be induced by A.hydrophila in vivo and in vitro, while its m RNA transcription was also influenced by stimulation with LPS or flagellin in vitro. The over-expression of Ci Cdc42 could basically lead to up-regulation of Ci IL1-β, Ci TNF-α and Ci IFN but not Ci PAK1 by the induction of Ci Cdc42 and bacterial PAMPs, while Ci Cdc42 knockdown decreased the transcriptional levels of these three genes after challenge with these pathogens. The overexpression of Ci Cdc42 reduced bacterial invasion, while the suppression of gc Rac1 facilitated it. These findings demonstrate that Ci Cdc42 is involved in the grass carp host defense against pathogen stimulation and provide a novel prospective into the vital role of Ci Cdc42 in innate immunity. |
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