Cloning And Expression Analysis Of TLR Genes Of Mink And Raccoon Dog And Transcriptomics Studies Of PBMCs Of Raccon Dog

In recent years, with the development of breeding industry of fur-bearing animals, certain diseases broke out in some areas, duing to the improper management and the decline of disease resistance, had caused massive deaths. Consequently, in order to resolve the problems of sustainable development of fur-bearing animals’ breeding industry, it is an inevitable way to further study on animals’ immune defense mechanism, to explore the effective ways to improve body resistance, and to cultivate disease-resistant strains.Four pairs of primers were designed according to ferret TLR sequences in GeneBank to obtain the sequences of TLR4、TLR6、TRL7 and TLR8 genes of mink and then bioinformatics analysis of these sequences were performed. Furthermore, the expression of TLR4 and TLR7 genes in various tissues(Liver、Lung、Muscle、kidney、small intestine、spleen)and PBMCs in young and adult minks were analyzed by semiquantitative RT-PCR. Sequence homology analysis showed that there were higher similarities of mink when compared with carnivora(ferrets, polar bear, panda, walrus, seals, dog, tiger and cat). However, they were lower compared with avians and fish. Tissue expression analysis showed that TRL4 and TRL7 were widely and differently expressed in variety tissues of mink. Interestingly, the expression of TLR4 and TLR7 in young mink were much higher than that in adult mink.This was the first time in China to clone the full length of TLR8 by RACE and RT-PCR techniques, primers were designed according to the canine TLR8 gene sequences in GeneBank, the putative amino acid sequence and the gene were analyzed by bioinformatics software. The result showed that full length cDNA sequence was 3 199 bp, the ORF sequence was 3 114 bp in length, which encode a deduced protein including 1 038 amino acid residues. Blast research revealed 99.3% similarities between canis lupus familiaris and raccoon dog, and 89.2% to 92.9%similarities compared with other carnivore. Compared with fish, the similarities were far from similar.The next generation sequencing technology was implemented in RNA sequencing, de novo splicing, and information comparison of the peripheral blood mononuclear cells transcriptome. The results showed that, the transcriptome sequencing had received a total of 3.1 GB raw data and 32 245 804 reads. The number of reads after removing the informations of carriers were 28 797 350. Using the TransDecoder to identify the coding regions of the assembled transcripts after the quality controllment and the de novo splicing. There were a number of 118 868 raccoon dog transcripts with the average length of 525.53 bp. Functional annotation of ORF and contigs using Trinotate, and prediction of sequence information using Uniprot database, RNAMMER, eggNOG, GO, KEGG showed that, B cell or T cell receptor signaling pathway, Toll-Like, NOD-Like or RIG-Like receptor signaling pathway,Phagosome pathways and Cytotoxic pathways mediated by NKT cell of KEGG annotation, as well as signal transduction mechanisms and defense mechanisms of COG involved in immune response and disease-resistant activities of raccoon dog.The purpose of this study was to discuss the important immune molecules and immunological mechanisms of fur-bearing animals(mink and raccoon dog) by sequencing, bioinformatics analysis and tissue distribution studies of TLR4, TLR6, TLR7 and TLR8 genes of mink, as well as by the cloning, sequencing, analysis of the predicted protein of the full length of TLR8 gene of the raccoon dog. Furthermore, the next generation sequencing technology was implemented in RNA sequencing, and information comparison of the peripheral blood leukocyte transcriptome of raccoon dog so as to provide the foundation and reference of further study of diseases prevention and immunologic mechanism, and to accumulate the genetic material for cultivation of anti-disease varieties.