Cloning And Expression Analysis Of TLR22&Relish Genes In Penaeus Monodon

Black tiger shrimp (Penaeus monodon) is an important economic marineaquaculture species. In recent years, outbreaks of diseases, degradation of germplasmresources and environmental pollution leading to high mortality during breedingprocess, resulting in huge economic losses，They have become the bottleneck of thesustainable and healthy development of the shrimp farming industry. In order toscreen immune-related genes, Improve breeds of Black tiger shrimp at the geneticlevel, improve disease resistance ability of Black tiger shrimp we explored theimmune-related genes and analysis their regulatory mechanism from the immunesystem. In this study, we cloned immune-related functional genes TLR22and Relish,and explored their the expression and regulation mechanism. At the same time, theeffect of Relish on Black tiger shrimp during development was also studied.TLR22is one important member of Toll-like receptor family. Toll-like receptor isthe greatest found of the host innate immune system since the20th century. Toll isbelong to pattern recognition receptor (PRR), through the extracellular region of theLRR domain specific recognition of pathogen associated molecular (pathogenassociated molecular pattern, PAMP), initiate the intracellular signal transductionpathways, induction of interferon, inflammatory factors, and other anti-inflammatorycytokines. Obtained PmTLR22gene from the black tiger shrimp is a partial cDNAwhich is3680bp long with3045bp open reading frame (ORF) encoding1014aminoacids. PmTLR22protein contains extracellular region, transmembrane andintracellular region. Its extracellular region contains12leucine repeat (LRRs) andintracellular contains a relatively conserved Toll/interleukin1receptor domain(Toll/IL-1R, TIR). PmTLR22protein‘s LRRs part in the extracellular is highlyconserved at the4th leucine residues (L).TLR22is near to TLR3in evolution. Real-time quantitative PCR shows that PmTLR22expression is relatively high in thehepatopancreas, followed by the intestines and stomach in the detection11kinds oforganizations. Using Real-time quantitative PCR detection the PmTLR22inhepatopancreas at different immune-stimulating conditions, the expression resultsshowed that TLR22in hepatopancreas can be induced by WSSV and Vibrio vulnificusand not by Staphylococcus aureus.24h after WSSV infection PmTLR22expressionwas upregulated23-fold, after6h and9h Vibrio vulnificus infection PmTLR22expression was significantly upregulated. It proved that the virus nucleic acidanalogue poly (I:C) and R484can activated the expression of PmTLR22, indicate thatPmTLR22can be induced by virus PAMP, PmTLR22may be an mode recognitionmolecules of PAMP. The recombinant fusion protein of PmTLR22extracellulardomain, and its weight is about58kDa, provide a basis for further functional study.Relish is one member of the NF-κB family. Rel/NF-kB transcription factors playcentral roles in many signal transduction pathway, which regulates a variety ofphysiological and pathological processes including innate immune response,inflammatory response, cellular proliferation and differentiation, and apoptosis. Herewe cloned a Relish homologue, PmRelish, from the constructed hepatopancreascDNA library of Penaeus monodon. The full-length cDNA of PmRelish is5112bpwith an open reading frame of3561bp that encodes1186amino acids. PmRelishprotein contains the typical architecture of NF-κB family member, including aconserved N-terminal Rel homology domain(RHD), a nucleus localization signal, sixC-terminal ankyrin repeats and a death domain, suggesting that it belongs to the classI NF-kB. PmRelish is constitutively expressed in the detected tissues, with highexpression in hemocyte. Using the real time PCR, we analyzed the expression ofPmRelish after challenged with different stimulus. The result showed that PmRelishin hepatopancreas can be induced. Meanwhile PmRelsih is regulated by Vibriovulnificus, Staphyloccocus aureus, and WSSV, and obviously unregulated by Vibriovulnificus. Furthermore, we confirmed the viral nucleic acid analogue poly(I:C) andR484activate the expression of PmRelish. In conclusion, our research reveals Relishgene is an important immune factor in tiger shrimp, and directly or indirectly involvedin the immune response against bacterial and virus. Besides, the expression ofPmRelish in the development process of the black tiger shrimp is as follows: Theexpression of PmRelish among four periods of larval development is of significantdifference, with the highest expression level in mysis stage. The expression profile of PmRelish at different developmental stages in testis is similar to that in ovary, with alow expression level at immature and post-mature phases. The expression level ofPmRelish is highest in testis of male tiger shrimp when body length is about10-12cm,and is highest in mature stage ovary of female tiger shrimp with a14.3folds higherthan that in oogonium stage. In conclusion, our research reveals that PmRelish mayplay an important role in regulating the larval, gonad and embryonic developmentstage of the black tiger shrimp.These results revealed that PmRelish may also play animportant role in the development process of the black tiger shrimp.