Molecular Cloning And Expression Analysis Of The TLR18,TLR19 And TLR21 Genes In Yellow Catfish(Pelteobagrus Fulvidraco)

Yellow catfish(Pelteobagrus fulvidraco)is an important commercial fish in China.With the rapid development of intensive farming in recent years,the yellow catfish culture industry has been severely affected by bacterial diseases which have led to heavy economic losses every year.The innate immune system is primarily important for fishes to respond against bacte rial diseases due to the evolutionary status and poikilothermic nature of fish.As one type of PRRs in the innate immune system of vertebrates,TLRs play important roles in recognizing specific PAMPs and triggering distinct antipathogen immune responses.Therefore,understanding the innate immune defence mechanisms of TLR genes may contribute to developing healthy techniques for disease control and enhancing the development of yellow catfish aquaculture.In this study,we aim to:(1)clone partial or complete cDN A sequences of the Pf_TLR18,Pf_TLR19 and Pf_TLR21 genes from yellow catfish and analyze their molecular characterization;(2)detect the spatial and temporal expression patterns of the three genes using real-time quantitative PCR;(3)investigate the mRNA expression changes of the three TLR genes after challenge with Aeromonas hydrophila;(4)investigate the mRNA expression changes of the three TLR genes after challenge with LPS,PGN and Poly(I:C)in isolated peripheral blood lymphocytes;and(5)prokaryotic expressions of the three genes’ extracellular domains.The main results are as follows:1.Characterization of Pf_TLR18,Pf_TLR19 and Pf_TLR21 cDNAsIn this study,partial c DNA sequences of the Pf_TLR18 and Pf_TLR19 genes and complete cDNA sequence of the Pf_TLR21 gene were cloned from yellow catfish(Pelteobagrus fulvidraco).The open reading frames(ORFs)of the Pf_TLR18,Pf_TLR19 and Pf_TLR21 genes were 1956 bp,2262 bp and 2949 bp in length,encoding 651,753 and 982 amino acids,respectively.Homologous identity revealed that the Pf_TLR18,Pf_TLR19 and Pf_TLR21 genes have high nucleotide and protein sequence similarities with those of channel catfish,2.Quantitative analyses of mRNA expression levels in adult tissues and early developmental stagesThe Pf_TLR18,Pf_TLR19 and Pf_TLR21 genes were expressed in all tested tissues,all three genes had their highest relative expressions in the spleen than other tissues.Expressions of the three TLR genes were very high from the fertilized egg stage to the late blastula stage.Subsequently,the expressions of the three genes were decreased significantly to a relative low level and were maintained to newly hatched larvae stage.Then the expressions of the three genes exhibited an up-regulated trend.3.Quantitative analysis of mRN A expression levels in five tissues after A.hydrophila challengeThe expressions of all three genes were significantly up-regulated from 72 to 168 hpi,after a fluctuation from 12 to 24 hpi in blood.The expression level of Pf_TLR18 mRN A in the spleen was up-regulated from 12 to 120 h post injection(hpi),and reached a peak value at 72 hpi.In the head kidney,it was significantly up-regulated from 6 to 48 hpi,with a peak value at 48 hpi.In the trunk kidney,it was significantly up-regulated at 6 hpi and reached its highest value at 24 hpi.In the liver,it was significantly up-regulated at 6 hpi and reached a peak value at 12 hpi.The expressions of Pf_TLR19 mRNA in the spleen and head kidney were significantly up-regulated from 48 to 168 hpi and 24 to 48 hpi,respectively.In the trunk kidney,it was significantly up-regulated at 24 hpi and reached a peak value at 48 hpi.In the liver,it was significantly up-regulated from 120 to 168 hpi.The expression level of Pf_TLR21 mRNA in the spleen,head kidney,trunk kidney and liver were all significantly up-regulated after a down-regulation at 6 and 12 hpi.It was up-regulated from 24 to 168 hpi,and reached its peak value at 120 hpi in the spleen.In the head kidney,it was significantly up-regulated from 24 to 168 hpi,with a peak value at 24 hpi.In the trunk kidney,it was significantly up-regulated from 24 to 48 hpi and reached its peak value at 48 hpi.In the liver,it was significantly up-regulated from 120 to 168 hpi with a peak value at 120 hpi.4.Quantitative analysis of mRNA expression levels in in isolated peripheral blood lymphocytes after immune challengeFor LPS stimulation,the expression level of the three genes were significantly up-regulated at 3 h and maintained to 12 h.After PGN stimulation,the expression levels of the three genes were up-regulated at 3 h.Subsequently,the expressions of three genes exhibited a small decrease at 6 h,then decreasd to the control level at 12 h.For Poly(I:C)stimulation,the expression levels of three genes continually up-regulated from 3 h to 6 h,and reached a peak value at 6 h,then they decreased to the control level at 12 h.5.Prokaryotic expressions of the extracellular domainsThe pGEX-LRR18,p GEX-LRR19 and pGEX-LRR21 vectors have been successfully transformed into the Ecoli.BL21(DE3)and expressed by the chemical inducer IPTG,and the recombinant proteins mainly exist as inclusion.