Cloning,structure And Evolution Of TLR2/TLR4 And The Key MyD88 Gene In The Associated Downstream Signal Pathway In Zhangixalus Dennysi

Amphibians are one of the evolutionarily significant vertebrates,and they are important for their transition from water to land.About 350 million year ago,the ancestor of amphibians began to land,to adapt to the terrestrial environments,and evolve into different groups.The wet and uncovered skin of amphibians made them vulnerable to the pathogens,and immune adaptation is the key question they face during their origin and diversification.Due to their low position in the vertebrate history,the acquired immune system was not perfect and the innate immune system plays important roles during their response to the exposure and pathogens.Toll-like receptors are one of the important pattern molecular receptors,and they are the key bridges between the innate and acquired immune system.Zhangixalus dennysi belongs to the genus Zhangixalus in the family Rhacophoridae,order Anura,Class Amphibia.Due to their special arboreal characteristic,Z.dennysi is widely investigated by the evolutionist.However,to date,the types,structure and evolution of TLRs in Z.dennysi are still unknown.In this study,we obtained the brain transcriptome of Z.dennysi based on the R-seq sequencing,de novo assembly and function annotation were addressed.We used RACE to clone c DNA sequences of TLR2,TLR4 and My D88 in Z.dennysi and a series of bioinformatics analysis were conducted to reveal the composition of amino acids,structure domain and characteristics of the protein.In addition,gene trees of TLR2,TLR4 and My D88 in representative vertebrates were reconstructed based on ML and BI methods.The main results are shown as follows:(1)We first obtained the brain transcriptome of Z.dennysi,which includes 35244 unigenes with the length above 500 bp,19123 unigenes with the length above 1000 bp.Total of 18711 unigenes were annotated according to SWISSPORT,13878 unigenes were for KOG annotation,and 16810 unigenes were for GO annotation.Most of the unigenes were included in the different biological function and signal pathway.Our study is the first transcriptome study on Z.dennysi,and the data obtained here will provide insights for the taxonomy,biochemical and adaptive evolution for Z.dennysi in the future.(2)We first cloned the Open Reading Frame(ORF)of the c DNA of TLR2,TLR4 and My D88 in Z.dennysi.The length of ORF of TLR2 for Z.dennysi is 2362 bp and it encodes a protein with 786 amino acids.The weight of the predicted TLR2 protein is 89.9k D,and the PI is 5.83.It includes three domains,i.e.,outer,trans-membrane,and inner cellular domains,and it also have one signal peptide,one trans-membrane,one lower complicated domain,9 LRR and one TIR.The ORF of TLR4 for Z.dennysi lacks a few amino acids in the 5' end,the obtained ORF is 2287 bp in length with 761 amino acids encoded.It includes 6 LRR,one lower complicated domain,one LRR TYP,one LRRCT,one trans-membrane and one TIR domain.The compete ORF of My D88 in Z.dennysi was obtained.It is 862 bp in length and encodes a protein with 286 amino acids,weight of 32.8 k D,and PI of 5.83.It includes one DEATH domain and one TIR domain.(3)Analysis of the phylogenetic relationships of vertebrate TLR2,TLR4 and My D88 were conducted.A series of variations occurred in different vertebrate groups for TLR2 and TLR4,whereas the My D88 appeared to be more conserved.Our results also suggested the adaptive variations might occurre for TLR2,TLR4 and My D88 genes in amphibians and different vertebrate groups,and the important roles of TLR2,TLR4 and My D88 in the immune adaptation for amphibians during their transition from water to land.