A Preliminary Study Of Transcriptome In Mandarin Fish (Siniperca Chuasti) Under The PolyI:C Or LPS Stimulation And The Cloning And Expression Research Of Its Two Toll-like Receptor Genes

Siniperca chuatsi,belonging to Perciformes,Sinipercinae and Siniperca,is one kind of the most famous valuable fish and the important economic species living in freshwater of China.Due to the expansion of breeding,fish drug abuse and the germplasm degradation,the diseases of mandarin fish are rapidly increasing,which is going to be a trend of outbreak.Every year,it raises the risk of farming and brings seriously economic losses.One of the most effective ways of reducing the harm is to start the research of disease-resistant breeding on mandarin fish through the selection of fish disease-resistant genes.At first,we took the strategy of comparative transcriptome sequencing to select a variety of immune pathways with their different expression genes from the data to understand the disease-resistant mechanism preliminarily.We also carried out the clonings and sequences analysis of TLR3 and TLR7 genes,which are two members of the TLR families of Siniperca chuatsi.We hope bring a certain basis of diging molecular markers about the TLR signal pathway and the answering mode of resisting the invasion of pathogen on the downstream key genes in mandarin fish under the Poly I:C or LPS stimulation.The results are as follows:1.We built three librarys of the transcriptome for Siniperca chuatsi under the stimulation of Poly I:C,LPS and PBS,respectively.The clean data within these three groups reached 7.08 Gb,and 23.77 Gb clean data was obtained in all.209,854 transcripts and 87,628 unigenes were obtained though sequencing of denovo assembly.And the average length of them is 1833.16 bp and 944.25 bp,with an average GC content of 48.72 %.The Q30 of every sample was not less than 91 %.KEGG classification and the enrichment of DEG analysis showed many immune related pathways such as JAK-STAT,TLR,NOD,RIG and their differentially expressed genes,such as EP300,JIP-1,IL-1β and IL-8 were involved in these immune function of resistance.The results above provided a valuable clue for further research of immune mechanism in mandarin fish.In addition,we also developed some interferon genes,inflammatory factor family genes and other immune adjustment factors,that will provide a molecular basis for the study of immune response of mandarin fish against different pathogens.2.We took the way of RACE to clone the TLR3 and TLR7 Genes of Siniperca chuatsi.The bioinformatics analysis results shows that the lengths of their c DNA are 3181 bp and 3408 bp,respectively.Each of them has an ORF of 2769 bp and 3156 bp,and encodes 922 and 1051 amino acids respectively.Both of them have typical structures of rich repeated leucine domain of TLRs.The amino acid composition analysis found that the hydrophobic amino acid accounted for 43.0 % of the protein encoded by the TLR3’s c DNA,and the hydrophilic amino acids accounted for 51.4 %.The hydrophobic amino acid of TLR7’s was 39.7 %,and hydrophilic amino acid was 55.8 %.The homologous comparison of amino acid showed that both of them are most similar to that of the perciformes.TLR3 and TLR7 of Siniperca chuatsi contain 3 and 1 introns,respectively.The exon 2 and 3 of TLR3 and exon 2 of TLR7 are similar to that of the rest of species in this experiment.3.To measure the expressions in health different tissues,the q PCR specific primers were designed according to the sequences of Siniperca chuatsi TLR3 and TLR7 c DNA.The results showed:TLR3 owned the highest expression in the liver,at 15.35;the level in the intestine was second(8.85);and merely expressed in the rest tissues;and TLR7 owned the highest expressions in the spleen and the head kidney,94.02 and 87.93,respectively.The second was 29.41 in the muscle;and merely expressed in the other tissues.Therefore,the expressions of Siniperca chuatsi TLR3 and TLR7 are all tissue-specific patterns.4.The expressions of the related genes on the TLR7-Myd88 pathway have shown that: after the injection of Poly I:C,TLR7 and Myd88 in the head kidney were significantly up-regulated at 3 h(P<0.01)respectively,and IRAK1 was up-regulated very markedly at 48 h(P<0.01).The locations of these two downstream signaling molecules of TLR7,may result in the sequential activation on this pathway.In the spleen,with the same stimulation,Myd88 was significantly increased at 6 h(P<0.01),which could be involved in the immune response under the Poly I:C stimulation.IRF7 and IFN-α3 were significantly up-regulated in the spleen at 6 h after the Poly I:C stimulation(P<0.05),but the expression of IRF7,which inducing the secretion of interferon,was not markedly up-regulated.So the uptrend of IFN-α3 may also indirectly affected by other major genes on the TLR3-TAK1 pathway.Under the LPS stimulation,TLR7 in the spleen was significantly up-regulated at 3 h(P<0.05)and it was significantly induced at 12 h in the head kidney(P<0.01),which indicating that TLR7 could response to LPS.IRAK1 was significantly up-regulated in the spleen among 3-48 h point after the LPS stimulation,which may caused by other members of TLRs subfamilies,besides TLR7.Myd88 was significantly induced in the spleen after the LPS stimulation at 3 h and 6 h,it may show that Myd88 was involved in the response of LPS.IRF7 was significantly induced to the peak at 3 h in the spleen(P<0.01).Moreover,IFN-α3 increased markedly at 24 h(P<0.05).So,LPS may cause the immune response of spleen and activate the expression of interferon related genes to suppress the multiplication of pathogeny through promoting the secretion of interferons.The expressions of the related genes on the TLR3-TRIF pathways have shown that: under the stimulation of Poly I:C,the expression of TLR3 in the head kidney become markedly higher at 3 h(P﹤0.01),it is may because TLR3 acts as a receptor of ds RNA in virus,which could be induced rapidly by Poly I:C.In spleen,TLR3 was significantly increased until 24 h after the Poly I:C.Head kidney always plays a role of a central and a peripheral immune organ.But spleen usually acts as a peripheral one,which may cause the response to Poly I:C lagging behind.In the head kidney,TLR3 and TRAF6 significantly reached their peaks at 3 h(P﹤0.01),and TAK1 was significantly induced until 48 h(P﹤0.01).It is shown that the locations of these two downstream signaling molecules of TLR3 may result in the sequential activation on the pathway.Under the LPS,the expression of TLR3 in the spleen was significantly lower than that in the control group at 3 h(P<0.01).We speculated that TLR3 couldn’t be a receptor of LPS.TAK1 showed a increase at 3 h in the head kidney after the Poly I:C(P<0.01)and ikkβ was also up-regulated at the same time(P<0.05),with IL-8 and IFN-α3 also markedly rising up at 3 h.So IL-8 and IFN-α3 could be induced by TAK1 and ikkβ.