Functional Studies On The Regulation Of Immunity In Chinese Shrimp, Fenneropenaeus Hinensis By The Nuclear Transcription Factor, NF-κB Family Genes

Nuclear transcription factors NF-KB family genes control several importantpathways in the innate immunity of invertebrate by regulating the transcription ofimmune effectors to defense against foreign pathogen infections. In this study, theregulating function of NF-κB transcription factors in the immunity of shrimp wasstudied and some progresses were obtained:1. A new complementary DNA (cDNA) isoform of Relish gene designated asFcRelish2was cloned by rapid-amplification of cDNA ends (RACE) approach.Compared with the reported FcRelish cDNA previously, the deduced amino acidsequence of FcRelish2contained not only the entire Rel homology domain (RHD)and nucleus localization signal (NLS), but also the ankyrin repeats (ANKs), deathdomain (DD) and caspase cleavage site. Multiple alignment and phylogenetic analysisindicated that FcRelish2showed high similarities with Relish homologues of otherarthropods. Through analysis on the gene structure of Relish, we found that FcRelishand FcRelish2were the spliced isoforms of Relish gene. The promoter sequence andtranscription factor binding sites of FcRelish gene were analyzed.2. The transcriptional expressions of different antimicrobial peptides (AMPs),including Penaeidin3(Pen3), Penaeidin5(Pen5), crustin, and anti-lipopolysaccharidefactor (ALF) were detected by real-time reverse transcription-polymerase chainreaction (RT-PCR) when shrimp were injected by different types of bacteria(Micrococcus lysodeikticus, ML, G+and Vibrio anguillarium, VA, G-). Different AMPs showed different expression profiles when shrimp were injected with one typeof bacteria, and each AMP also showed different expression profiles when shrimpwere challenged by different types of bacteria. The transcriptional expression ofRelish gene could be modulated by the injections of bacteria or white spot syndromevirus (WSSV), and Relish could regulate the transcription of different AMPs.3. The transcriptional expression of Dorsal gene could be also modulated by theinjections of bacteria or WSSV. The response of Relish gene to G-bacteria might befaster than that of Dorsal gene. Dorsal gene could also regulate the transcription ofdifferent AMPs.4. A cDNA of the inhibitor protein of NF-κB (IκB) named as FcCactus was firstcloned from Chinese shrimp. The deduced amino acid sequence of FcCactuscontained the key features of IκB proteins, and showed high similarities with IκBhomologues from other seven arthropods. The transcripts of FcCactus gene wereubiquitously expressed in all detected tissues and the expression level was the highestin the muscle, hemocytes, heart and lymphoid organ. The transcriptional expression ofFcCactus gene could be changed by the injections of bacteria or WSSV, and FcCactuscould regulate the transcription of different AMPs and antiviral factor (AV).5. In order to study the functional relationships of Relish, Dorsal and Cactus, RNAinterference (RNAi) approach was used to silence the expression of them. The resultsindicated that the transcriptional expression of Relish2was down-regulated and thetranscriptional expression of Cactus was up-regulated when FcDorsal was silenced..The transcriptional expression of FcCactus was up-regulated, while the transcriptionalexpression of Dorsal showed no variation when FcRelish was silenced. Thetranscriptional expressions of both FcRelish and FcDorsal were up-regulated whenFcCactus was silenced. It could be inferred that the transcriptional expression ofRelish gene was regulated by Dorsal gene, and there existed functional relationshipsamong Cactus, Dorsal, and Relish genes in shrimp.6. For the fisrt time by combining RNAi approach with SSH approach, the forward and reverse subtracted libraries were constructed between Relish silencedshrimp and normal shrimp injected with Vibrio for1h by suppression subtractivehybridization (SSH) and genes regulated by Relish were screened. In the forwardlibrary,43unique genes were identified and in the reverse library,57genes wereidentified. Twelve differentially expressed genes were selected to be confirmed bysemi-quantitative RT-PCR. Genes encoding Early cuticle protein1(ECP1), Earlycuticle protein5(ECP5), Toll-like receptor protein (TLRP), antiviral factor (AV),C-type lectin receptor (CLR), and Thrombospondin (TSP) showed down-regulatedexpressions in the shrimp challenged with VA after Relish gene was silenced, whilegenes encoding S-adenosylmethionine synthetase (SAMS), Carcinolectin5a isoform(CL5a), and Carcinolectin5b-5(CL5b-5) showed higher expressions on the contrary.The informations are important for us to understand the role of Relish in shrimpimmunity.