The Preliminary Studies On The Gene Structure,Expression Profiles And Characterization Of Chinese Giant Salamander (Andrias Davidian) TLR7 And MyD88

Toll-like receptors(TLRs),as important members in the innate immune system,represent the first line of defense mechanisms against the microbial pathogens invading.After recognizing pathogen-associated molecular pattens(PAMP),via myeloid differentiation factor88(My D88)dependent or TIR domain-containing adaptor-inducing interferonβ(TRIF)dependent signaling pathway,TLR activates the transcription factors NF-κB and IRF,and initiates the production of inflammatory cytokines.Currently,TLR family members have been found in human,mouse,xenopus,zebrafish,etc.The Chinese giant salamander(Andrias davidianus),called Wawa-yu,which is unique and rare amphibian in china and is the largest extant amphibian species in the world.As an ancestral organism,the Chinese giant salamander represents the transition from aquatic to terrestrial life,and it is considered to be an important model in scientific research of the phylogeny,biodiversity,and the earth’s evolutionary history.However,in recent years,with the rapid development of artificial breeding and culture of giant salamander,disease problems have become grate concern,in which the Chinese giant salamander iridovirus(GSIV)infection caused huge economic losses.Therefore,the studies on Chinese giant salamander pathogen infection and host defense mechanisms,are of not only important scientific significance,but also have a wide-range application in practice.However,compared to other aquatic organisms,there is limited information of the immune system and molecular immunology on Chinese giant salamander.In this study the full-length c DNAs of Chinese giant salamander TLR7(Cgs TLR7)and My D88(Cgs My D88)genes were cloned using RACE-PCR.The structures and functions of Cgs TLR7 and Cgs My D88 were analyzed by bio-informatic method.The expression profiles of the genes in the tissues of normal Chinese giant salamander were examined,and the immune responses post GSIV infection were also determined by real-time PCR.In addition,the localization of Cgs My D88 protein in GS-M cell and the overexpression the Chinese giant salamander represents induced NF-κB were studied.The results reported herein provide a fundamental base for the further study of immune evolution system,structure and function and anti-infection molecular mechanisms in Chinese giant salamander.The results are as follows:1.Based on the template of total RNA extracted from the Chinese giant salamander kidney,the full-length c DNA sequence of Cgs TLR7 and Cgs My D88 was cloned by RACE PCR.The full-length c DAN of Cgs TLR7 and Cgs My D88 genes were 3747 bp and 1423 bp respectively,and the length of ORF of the two genes were predicted as 3150 bp and 879 bp that encode 1049 aa and 292 aa respectively.The length of the 5’ UTR of the Cgs TLR7 and Cgs My D88 genes were 144 bp and 387 bp and the length of the 3’ UTR were 453 bp and 157 bp,respectively.The SMART program was used to analyze the structures of the proteins,the results,demonstrated that the Cgs TLR7 protein included an extracellular signal peptide,19 leucine-rich repeat(LRR)motifs and an intracellular Toll/interleukin-receptor domain.Cgs My D88 encodes a typical death domain at the N-terminus and a conservative Toll/IL-1R(TIR)domain at the C-terminus.The TIR domain of Cgs TLR7 and Cgs My D88 has three highly conserved regions box for signaling transduction,respectively.The BLASTp search revealed that the amino acid identity of Cgs TLR7 to turtle,frog and chicken was 71%,64% and 65%,respectively.The amino acid identity of Cgs My D88 shared highest homology with frog and turtle that both are 73%.The amino acid sequence of Cgs My D88 has 69% identity with chicken.Based on deduced amino acid sequences of Cgs TLR7 or Cgs My D88,the phylogenetic trees were constructed and showed similar results.The tree was divided into two branches.One branch was from piscine and the other branch was from mammals,avians,reptiles and amphibians.Chinese giant salamander and frog belonged to one branch and showed close to turtle.2.The q RT-PCR results indicated that the Cgs TLR7 and Cgs My D88 m RNA were detected in all selected organs.The highest expression of Cgs TLR7 was observed in the liver.The lowest expression of Cgs TLR7 was in intestine.Meanwhile,the highest expression of Cgs My D88 m RNA was observed in the kidney and the lowest expression was in the liver.After GSIV infection,the expression of Cgs TLR7 reached a peak in the kidney and spleen at 12 h and 48 h post-infection,respectively.The expression of Cgs My D88,was up-regulated at 6 h post-infection with GSIV and then decreased at 24 h,reached a peak at 48 h and decreased at 72 h,but the m RNA levels were still higher than those in control group.In kidney,m RNA expressions of Cgs My D88 was decreased at 6 h and reached a peak level at 48 h post-infection.Those results showed that Cgs My D88 plays an important role in the Chinese giant salamander innate immunity.3.Constructing the expression plasmid of pc DNA3.1(+)-My D88 and transfecting the Chinese giant salamander muscle cell line(GS-M),then the immunofluorescent analysis was conducted in this study with the anti-his antibody against GSIV.The results revealed that Cgs My D88 protein was most distributed in the GS-M cytoplasm and less in the nucleus.In order to verify whether Cgs My D88 able to activate the nuclear factor NF-κB,the pc DNA3.1(+)-My D88 plasmid,p NF-κB-Luc plasmids and p RL-TK plasmid were co-transfected into GS-M cells by luciferase reporter gene assay.Cgs My D88 induced NF-κB activity is approximately 1.88-fold higher than the control plasmid,suggesting that Cgs My D88 can activate the nuclear factor NF-κB activity downstream.