Genetic Polymorphism And Molecular Evolution Of Antibacterial Peptide DefensinB In Silkworm

Innate immunity of insect is composed of cellular immunity and humoral immunity.The synthesis and secretion of antimicrobial peptides is an important part of humoral immunity,which plays an important role in immune defense of insects.The evolution of antimicrobial peptides(AMPs)which is a kind of important immune effect molecules,has always been an focus of AMPs research.Silkworm,Bombyx mori,is an important economic insect and a representative of the research model of Lepidoptera.At present,there are 7 kinds of antibacterial peptide family,ie.,Cecropin,Moricin,Enbocin,Gloverin,Lebocin,Attacin and Defensin,in Silkworm.Defensin is a type of antibacterial peptide which is rich in cysteine.In the silkworm,the Defensin family has two members,namely DefensinA and DefensinB.DefensinA has been identified as an artificially selected target gene and has experienced a bottleneck effect,but the evolution of DefensinB is not yet reported.In our previous studies,we found that there was difference of DefensinB sequence among various varieties of silkworm.In the present study,DefensinB gene was amplified and sequenced from eighty domesticated silkworm strains and wild silkworm from six different places of China.Based on the nucleotide polymorphism analysis and neutral detection,the main results were as followed.1.Sequence of the DefensinB gene from 80 Bombyx mori strains showed that the DefensinB CDS length from Bombyx mori was 243 bp.The sequence was highly conserved and only several substitutions occurred in CDS.However,there were about 200 bp insertions or deletions due to different intron lengths.2.According to the length and nuclotide sequences,DefensinB was divided into two types,L-type(L1,L2 and L3)and S-type(S1,S2 and S3).Both L1 and L2 was exclusivehaplotype for tropical system.L3 is a haplotype shared by Japanese system,European system and tropical subtropical system.S1,S2 was an exclusive haplotype respectively for Chinese system and Japanese system.The haplotype network mediating map showed that the long and short types might be separated and evolved from ancestral genes independently.3.Nucleotide polymorphism analysis showed that the nucleotide polymorphism? of the DefensinB gene was reduced by 35% compared to the wild silkworm,and the population dynamic parameter ? was reduced by 75%,showing that the polymorphism of DefensinB gene was lost during the domestication of silkworm.4.The neutral detection showed that the total Tajima 's D value of the antibacterial peptide DefensinB gene from 80 varieties of silkworm was significantly different number of4.744(***p<0.001),indicating that DefensinB gene was under direction selection in domesticated silkworm.However,Tajima's D value of the wild silkworm is 0.08767,which was not significant,showing that DefensinB gene was under balancing selection in wild silkworm.All these results implied that DefensinB gene of silkworm and wild silkworm had undergone a different evolutionary process.5.By constructing the phylogenetic tree of the DefensinB gene,it can be found that the long and short varieties form two distinct branches.In the short-type branches,S1 and S2 were located in one sub-branch,and S3 forms another sub-branch.In the long-type branch,L1 and L2 were located in one sub-branch,and L3 forms another sub-branch.In addition,the wild silkworms of Jiujiang and Nanchang cluserted together and the rest were scattered and distributed in the evolutionary tree.6.The overall selection pressure of the DefensinB gene in the silkworm was purifying selection.No positive selection site was found.In summary,the nucleotide polymorphism of DefensinB gene of silkworm was lower than that of wild silkworm.DefensinB gene was under the pressure of purifying selection in domesticated silkworm rather than balancing selection in wild silkworm.