Research On The Functions Of Relish, Toll And Pellino In The Innate Immunity Of Procambarus Clarkii

Procambarus clarkii,commonly known as red crayfish or small lobster,was introduced into China in the 1930 s.With its strong adaptability and growth ability,P.clarkii has rapidly occupied an important position in freshwater aquaculture industry for decades.It has become one of the most valuable shrimps in the world because its delicious taste and high nutritional value in recent years,with the increasing demand for P.clarkii,the scale of culture is also expanding.The disease of P.clarkii caused by various pathogenic microorganisms comes with it,and the culture industry is deeply disturbed by it.P.clarkii are invertebrates,lack of adaptive immune system,only rely on innate immunity to resist the invasion of pathogenic microorganisms.Therefore,the research on innate immunity of P.clarkii is particularly important.In this study,we studied the immune function of Relish,Toll and Pellino in P.clarkii.1.Nuclear factor NF-?B plays a key role in the innate immunity of invertebrates,Relish is one of the members of NF-?B family.Two Relish subtypes(SPc Relish and LPc Relish)were identified from P.clarkii and their functions were analyzed.The long subtype LPc Relish is completely spliced,with 25 exons and 24 introns,with a total length of 1187 bp;the short subtype SPc Relish is alternatively spliced,with a total length of 418 bp,including the retention of exon 1-9 and intron 9.LPc Relish contains Rel homologous domain(RHD),Ig like domain,plexin,IPT domain and ankyrin repeat(ANK)inhibitory domain.However,SPc Relish only contains RHD and IPT domains,and there is no ANK domain.Vibrio parahaemolyticus can transcribe SPc Relish and LPc Relish.After SPc Relish gene was interfered and V.parahaemolyticus was used to stimulate shrimp,it was found that the expression of some antimicrobial peptides was inhibited,such as ALF-7032,ALF-13162 and Crustin-42012,while the expression of ALF-41125,ALF-42430,Crustin-41354 and Crustin-42993 increased significantly.After the LPc Relish gene was interfered,the shrimp was stimulated by V.parahaemolyticus.It was found that all the antimicrobial peptides except ALF-41125 were inhibited.According to these results,we believe that these two Relish subtypes play important roles in the immune response of P.clarkii to invasion.2.Three new Toll genes(Pc Tolla?Pc Tollb?Pc Tollc)were identified in P.clarkii.QRT-PCR analysis showed that Pc Tolla?Pc Tollb and Pc Tollc were widely distributed in all tissues of P.clarkii,and the highest expression level was found in intestine bothmale and female tissues.After WSSV stimulation,the expression of Pc Tolla?Pc Tollb?Pc Tollc in the intestine was significantly up-regulated.After Pc Tolla?Pc Tollb and Pc Tollc gene were interfered,WSSV was used to stimulate the shrimp.The results showed that Pc Tolla could regulate the expression of ALF-7032,ALF-9228,ALF-42430,Crustin-42012 and Crustin-42993 in the intestine of P.clarkii,Pc Tollb could regulate the expression of ALF-13162,ALF-9228,ALF-42430,Crustin-41354 and Crustin-42993,Pc Tollc could regulate the expression of ALF-13162,ALF-9228 and ALF-41125 The expression of,Crustin-42012,Crustin-41354 and Crustin-42993.This study shows that Pc Tolla?Pc Tollb and Pc Tollc genes of P.clarkii play important roles in the innate immune response induced by antimicrobial peptide expression.3.Pellino,as E3 ubiquitin ligase,also plays an indispensable role in the immune response of P.clarkii.A new pellino gene Pc Pellino was identified from P.clarkii.The total length of its c DNA is 1872 bp,including 1305 bp ORF,which encodes 434 amino acids,including low complexity region and Pfam: pellino domain.QRT PCR analysis showed that Pc Pellino was widely distributed in the tissues of P.clarkii,and its expression level was relatively high in gills and intestine;under the stimulation of V.parahaemolyticus,S.aureus and WSSV,the expression of Pc Pellino in gill and intestine was significantly increased.After Pc Pellino gene interference,shrimp were stimulated with V.parahaemolyticus,S.aureus and WSSV respectively.The results showed in gills that under the stimulation of V.parahaemolyticus,Pc Pellino gene knockout could significantly reduce the expression level of ALF-7032?ALF-9228?ALF-41125 ? Crustin-41354 and Crustin-42993,increase the expression level of ALF-13162;and under the stimulation of S.aureus,Pc Pellino gene knockout could significantly reduce the expression levels of ALF-13162?ALF-9228?ALF-42430 and Crustin-41354;the expression levels of ALF-7032 ? ALF-42430 ? ALF-41125 ?Crustin-41354 and Crustin-42993 were significantly reduced by Pc Pellino gene knockout under WSSV stimulation.In intestine,Pc Pellino gene knockout could significantly reduce the expression level of ALF-13162?ALF-42430 and ALF-41125,increase the expression level of Crustin-42012 under the stimulation of V.parahaemolyticus;and under the stimulation of S.aureus,Pc Pellino gene knockout could significantly reduce the expression levels of ALF-7032 ? ALF-42430 ?ALF-41125? Crustin-42012 and Crustin-41354;the expression levels of ALF-7032?ALF-9228?ALF-42430?ALF-41125 and Crustin-41354 were significantly reduced by Pc Pellino gene knockout under WSSV stimulation.These results suggest thatPc Pellino gene may be involved in the immune response of P.clarkii and play an important role in the process of ubiquitination.