Adaptive Evolution Of TLRs In Birds

Innate immunity,the first line of defense against pathogens,it plays an important roles in the invasion of pathogenic microorganisms in the early stage of immune response.Toll-like receptors(TLRs),as an important functional proteins that mediate innate immunity,directly interact with pathogens through pattern recognition receptors(PRRs)and regulate specific immune responses,is the bridge of innate immunity and acquired immunity.Because of the wide distribution,great differences in living environments and dietary diversity of birds,the viruses' types are also different.This research based on TLRs gene to discuss: 1)Phylogenetic relationship of the TLRs gene family of the avian? 2)Are the different evolution patterns of gene duplication of TLR1 and TLR2 subfamily? 3)What is the external factor to drive the evolution of birds TLRs?In this study,the TLRs gene of Lophophorus lhuysii,Tragopan temminckii,Chrysolophus pictus,Pavo cristatus,Phasianus colchicus and Coturnix coturnix was cloned,and combined with the existing data on the website,and the main results and conclusions of this thesis are as follows:1)In our study,we have obtained TLRs sequences from 6 species,its length ranges from 1959 bp(TLR1B)to 3180 bp(TLR7);The homology analysis of ten genes showed that they were extremely conserved,and the similarity between nucleotide and amino acid sequences were above 90%;Structure comparison revealed that the main difference being located in the extracellular leucine-rich repeat(LRR)region.2)The phylogenetic analysis shows that the entire TLRs gene family is divided 6 branches.TLR3 and TLR5 clustered a clade,TLR1 A,TLR1B,TLR2 A and TLR2 B clustered a clade,TLR4,TLR7,TLR15 and TLR21 clustered into a single clade separately.The evolutionary pattern of multiple copies of TLR1 and TLR2 subfamilies has branches,and TLR1 A is closer to TLR10 in mammals,which reveals functional differentiation,while TLR2 has no functional differentiation.3)The selective test implemented with Branch Model,Site Model and Branch-Site Model in PAML,and SLAC,FEL and FUBAR model in Datamonkey predicted that the evolution rate of bird TLRs was between 0.165(TLR21)and 0.4265(TLR5),which was under the strongly purification selection as a whole.However,some special sites were under the positive selected,a total of 126 common positively selected codons were identified by at least two methods,of which TLR5,TLR7 and TLR15 were most strongly positively selected.Most of these positive selected sites are located in the LRR domain in the extracellular region,suggested that these positive selected sites may have regulatory effects on genes.Through the analysis of the Branch-Site Model,it is showed that habitat environment and diet are the external factors driving the evolution of TLRs gene in birds.Habitat environment has a greater driving effect on TLR1 B,TLR2B,TLR3 and TLR4,but diet has a greater driving effect on TLR2 A,TLR2B,TLR15 and TLR21.It is revealed that TLRs gene have different selection pressures in the evolution of birds.In order to better adapt and resist the invasion of pathogenic microorganisms from different sources,which specific amino acid sites change of each TLRs locus generate some new function to provides a possibility for birds to adapt to different environments and feeding habits.In this study,we have obtained TLRs sequences from 6 species of Galliformes birds by using PCR amplification.In addition,we have combined with the TLRs gene of other vertebrates,and used various biology software to analysis the sequence characteristics,protein structure,phylogenetic evolution of TLRs gene family and evolutionary model of multi-copy genes in birds.Our study revealed that phylogenetic relationships,evolutionary patterns,environmental and dietary factors promoting affect of the avian TLRs gene family at the molecular level,which provided important basic data for the study of TLRs genes in birds and vertebrates.