The Cloning And Functional Analysis Of Immune Genes Defense And Apolipophorin-? In Actias Selene

Under the adverse external living environment insects can effectively resist the invasion of foreign bacteria,in addition to the protection of the body color and physical barriers such as Dryopteris,crustaceans and horny skin,insects also have to identify the body and different original Chuqing efficient immune system,to resist microbial and parasitic infections,thus it attached much attention to study the innate immune response mechanisms,which is to explain the origin of biological evolution,diversity and growth significantly.In this study,wild-type Actias selene(Lepidoptera: Saturniidae)for the study,immune system-related genes were cloned and characterized,as well as comparative and evolutionary studies,and explore their immune regulation induced modes and functions,in order to can We improve the the theoretical basis of lepidopteran innate immune defense mechanism,to provide a solid theoretical basis for subsequent effective use and develop applications.The main research contents and results are as follows:We isolated a novel defense gene(AsDef)cDNAs from Actias selene(Hubner 1806)(Lepidoptera: Saturniidae).It contained 691 nucleotides with a putative open reading frame(ORF)of 498 bp encoding 165 amino acid residues with a reeler domain.It showed significant similarities with defense proteins from several other lepidopteran insect species including Samia ricini(Donovan 1798),Antheraea mylitta(Drury 1773),Bombyx mori(Linnaeus 1758),Lonomia oblique(Walker 1855),Hyphantria cunea(Drury 1773)and Manduca sexta(Linnaeus 1763).The recombinant protein of As Def was expressed successfully in Escherichia coli(Migula 1895)cells,and the anti-AsDef antibody against rabbit was prepared.Results of real-time quantitative PCR(RT-qPCR)and western blot showed that ApDef gene was detected at a particularly high levels in fat body,while ApDef protein can only be detected in hemolymph.For immune functions,AsDef gene can be upregulated by E.coli,Micrococcus luteus(Cohn 1872),Beauveria bassiana(Bals 1912)and nuclear polyhedrosis virus(NPV),and showed a stronger sensitivity for gram positive bacteria than fungi and virus,while the least sensitivity for gram negative bacteria.Zone of inhibition experiments indicate the inhibition zone diameter of approximately the same,the time required shorter for gram-positive bacteria than negative bacteria These data indicated that AsDef plays an important role in innate immune responses of A.selene.Apolipophorin-?(ApoLp-?)is involved in lipid transport and innate immunity in insects.In this study,we isolated ApoLp-? gene(As-ApoLp-?)cDNA from Actias selene Hübner(Lepidoptera: Saturniidae).It contained 660 nucleotides with a putative open reading frame(ORF)of 561 bp encoding 186 amino acid residues.It showed similaritieswith ApoLp-? proteins from other insect species,especially Antheraea pernyi.The recombinant protein of As-ApoLp-? was expressed in Escherichia coli,and anti-As-ApoLp-? antibodies were prepared.The As-ApoLp-? gene was expressed at high levels in the fat body and integument,and at low levels in the hemolymph,testis,silk gland,malpighian tubule,ovary and midgut.As-ApoLp-? gene can be upregulated by E.coli,Micrococcus luteus,Beauveria bassiana and nuclear polyhedrosis virus(NPV),and showed a greater sensitivity for gram negative bacteria and fungi than for gram positive bacteria and virus.These data indicated that As-ApoLp-? may play an important role in innate immune responses of A.selene.By RNA interference experiments,ApoLp-? gene down-regulated directly or indirectly affect the expression of superoxide dismutase Cu/ZnSOD genes also down,there may be some interactive relationship among these important related genes in immune responses.