Comparison Of Structure And Function Of TLR7 Subfamily Genes Between Squaliobarbus Curriculus And Ctenopharyngodon Idellus

The haemorrhagic disease of grass carp is the key factor to restrict the green and safe culture of grass carp in China;the relationship between barbel chub(Squaliobarbus curiculus)and grass carp(Ctenopharygodon idellus)is relatively close,which belongs to the subfamily of arawanae.In recent years,our team found that barbel chub had stronger anti grass carp reovirus(GCRV)characteristics than grass carp,the study on the function of anti GCRV immune molecules can provide more material reference for grass carp disease resistance breeding.In this paper,the role of TLR7 subfamily(TLR7,TLR8 and TLR9)in GCRV resistance of barbel chub and grass carp was studied.The structural and functional differences of TLR7 subfamily genes between barbel chub and grass carp were studied by combining molecular biology,cell biology and bioinformatics,and the proteins interacting with TLR7 and TLR8 of barbel chub were screened.The main results are as follows:1 Comparative transcriptome analysis of the spleens of barbel chub and grass carpAfter GCRV challenge,the spleen tissues of barbel chub and grass carp were sequenced,and the original reads data of 37.50 G and 35.70 G were obtained.The results of comparative transcriptome analysis showed that there were 2307 differentially expressed genes in the spleen of barbel chub compared with grass carp after GCRV challenge,2054 of which were up-regulated genes and 253 of which were down-regulated genes.The results of GO annotation of differentially expressed genes and enrichment analysis of KEGG signaling pathway showed that the differentially expressed genes in spleen of barbel chub were mainly enriched in 56 go entries,such as cell process and single biological process,compared with grass carp,and the first 20 most credible enrichment KEGG signaling pathways included TLRs signaling pathway.The results of q PCR showed that the expression of TLR7 subfamily in the group of barbel chub and grass carp injected with GCRV was higher than that in the control group(injected with PBS of the same amount),suggesting that TLR7 subfamily participated in the anti GCRV immune response of barbel chub and grass carp.2 Comparative analysis of gene structure and expression pattern of TLR7 subfamily between barbel chub and grass carpThe full-length c DNAs of the TLR7 subfamily genes(ScTLR7,ScTLR8,and ScTLR9)of barbel chub are 3715 bp,4624 bp and 3687 bp,respectively,and the open reading frame(ORF)lengths are 3162 bp,3072 bp and 3180 bp,respectively,encoding 1053,1023 and1059 amino acids.Grass carp TLR7 subfamily genes(Ci TLR7,Ci TLR8,and Ci TLR9)c DNAs are 3354 bp,3766 bp,and 3468 bp in full length,and the open reading frame(ORF)length is 3156 bp,3072 bp,and 3177 bp,respectively,encoding 1051,1023 and 1058 amino acids.Barbel chub and grass carp TLR7 subfamily genes have typical structural features of TLRs family genes,consisting of N-terminal signal peptide,repeating leucine-rich domain(LRR),transmembrane domain(TM)and C-terminal Toll/interleukin-1 Receptor domain(TIR)composition;and it is highly expressed in immune tissues such as spleen.Barbel chub TLR7 and TLR8 contain the same number of LRRs as grass carp TLR7 and TLR8.Grass carp TLR9 contains one less LRR than barbel chub TLR9.There are amino acid differences in multiple LRR domains between barbel chub and grass carp TLR7 subfamily genes,and there are also amino acid differences in the signal-transmitting TIR domain.After GCRV infection,TLR7,TLR8 and TLR9 showed different trends in the spleen.The expression of TLR7 and TLR8 in the spleen of barbel chub peaked at 168 h,and the expression of TLR7 and TLR8 in the spleen was higher than that of grass carp.The trend of TLR9 expression in the spleen of grass carp decreased first and then increased at the initial stage of GCRV infection(6?24 h),which was different from the trend of TLR9 expression in the spleen of barbel chub(always decreasing).Myeloid differentiation primary response protein 88(My D88)in the spleens of barbel chub and grass carp 24 to 168 h after GCRV infection and interferon regulatory factor 7(IFN regulatory factor 7,IRF7)6 to 72 h after GCRV infection the expression trend is consistent,while the expression of type I IFN(I-IFN)in the spleen of barbel chub was higher than that in the spleen of grass carp at each time point.After GCRV infection,the transcription levels of ScTLR7,ScTLR8,ScMy D88 and ScI-IFN were up-regulated,and the expression levels of ScTLR7 and ScMy D88 were positively correlated.3 Comparative analysis of TLR7 and TLR8 gene functions of barbel chub and grass carpAfter interfering with TLR7 and TLR8 of grass carp and barbel chub,the expression changes of I-IFN and Myxovirus resistance(Mx)have different expression trends in grass carp and barbel chub cells,indicating that TLR7 and tlr8 play different roles in immune response to GCRV.After interference with barbel chub TLR8,the expression changes of I-IFN and Mx were up-regulated.This is similar to the results of the Su Jianguo team using sh RNA to interfere with Ci TLR8.It is inferred that ScTLR8 may play a negative regulatory role.The expression trend of I-IFN and Mx in TLR8 fusion plasmid cells fused with the LRR domain of barbel chub is more significant,indicating that the LRR domain of barbel chub TLR8 is more sensitive than the LRR domain of grass carp to recognize exogenous GCRV.The expression levels of I-IFN and Mx were lower in the cells transformed into the plasmid lacking the TIR domain than those transformed into the cells lacking the ScTLR8LRR-NT and LRR-CT plasmids,indicating that after the TIR domain deletion of barbel chub TLR8,the downstream resistance molecules immune expression is weakened,and the ScTLR8 TIR domain mainly plays a role in signal transduction.We Successfully constructed a barbel chub yeast two-hybrid nuclear system library,and screened the Snapin protein that interacts with barbel chub TLR8 protein.Barbel chub Snapin(ScSnapin)gene c DNA is 1000 bp in length,in which 5'untranslated region(UTR)is39 bp,open reading frame is 399 bp,encoding 132 amino acids,and 3'-UTR is 562 bp.The molecular weight of ScSnapin amino acid is deduced to be 149.77 k Da,and the isoelectric point is 9.77.The above results showed that TLR7 subfamily was involved in the GCRV immune response of barbel chub and grass carp.Because of the differences in LRR quantity,location and amino acid composition of LRR and TIR,TLR7 subfamily of barbel chub and grass carp showed different resistance differences.ScTLR7 and ScTLR8 of barbel chub play different roles in anti GCRV immunity.The established yeast two hybrid library of barbel chub will provide the basis for the follow-up research on the regulatory mechanism of related immune function genes of barbel chub.