Cloning, Expression And Analysis Of A Novel Defense Gene From Antheraea Pernyi

Immune reaction is generally divided into two branches,innate immunity and acquired immunity.Insects do not have perfect acquired immune system like mammal,therefore innate immunity becomes the main means of resistance to invading pathogens for insects.The innate immunity of insect can be divided into cellular immunity and humoral immunity,both of which work together to resist the infection of exogenous pathogens such as bacteria,fungi,virus.Cellular immunity includes phagocytosis,assembly and encapsulation type,while humoral immunity includes two mechanisms of forming melanin and producing antimicrobial peptides.Tussah is an important kind of wild silk insects which belongs to Lepidoptera,Saturniidae families,genera tussah,whose main distribution areas are China,India and South Korea.Its main value lies in that it can be employed as raw material of textile industry and it has high economic value such as medicinal and edible value,which can be used to supplement the essential amino acids human body needs.Therefore,research on innate immune defense-related genes of tussah has important theoretical significance for improving tussah silkworm's ability of defending disease.Besides,it is also important or promoting other related research on insects like Lepidoptera and it can provide a reference for prevention and cure of Lepidoptera pests.The thesis makes analysis of Antheraea pernyi defense genes by using molecular biology and bioinformatics and the main research results are the following:1.Cloning and sequence analysis of ApDefAccording to the EST sequence information of public database The full-length cDNA of ApDef is677 bp,with an open reading frame(ORF)of 492 bp that encodes a polypeptide of 163 residues.Nucleotide sequence analysis revealed that ApDef cDNA contains an 63 bp 5'-untranslated sequence,a122 bp 3 ?-untranslated region(3 ? UTR).ApDef contains a signal peptide of 18 residues(MMFAYIVAVVSALALTSA).The molecular weight of hemolin calculated from the deduced amino acid sequence is 17 kDa and a predicted isoelectric point(pI)of 4.74.ApDef is a kind of hydrophilic protein,contain a distinct signal peptide and no obvious across the membrane structure field was found.Subcellular localization show that ApDef is most likely located in the extracellular.Defenses from A.pernyi is most similar to H.cecropia than mammals,human and mice.2.Tissue distribution of ApDefTo test the tisssue-specific expression of Ap Def mRNA,the fat body,hemocytes,midgut,malpighian tubule and epidermis were sampled from fifth-instar larvae of A.pernyi.Total RNA of each sample was extracted from fifth-instar larvae of A.pernyi and cDNAs were synthesized from mRNAs with random hexamers using M-MLV reverse transcriptase(TAKARA,Japan).The experimental resultsshow that ApDef mRNA was specifically higher expressed in fat bodies t and very little in hemocytes and midgut.No ApDef mRNA was found in epidermis.To construct pET-28a-Defenses expression vectors,we employed PCR strategy to amplify Defenses cDNA encoding the mature protein from amino acids.The recombinant plasmids pET-28 aApDef were identified by sequencing,then transformed into competent E.coil BL21(DE3)cells for protein expression induced by different concentrations of IPTG.The recombinant proteins were analyzed with 12% SDS polyacrylamide gel electrophoresis(SDS-PAGE)and western blotting.In order to obtain polyclonal antibody,the purified fusion proteins were used to immunize a male New Zealand rabbits respectively.Fifth-instar larvae of A.pernyi were injected with heat-treated E.coli,Micrococcus luteus or Beauveri.Bassiana.The expression of ApDef mRNA in fat body was obviously induced and reached the top in 12 h.These data suggest that ApDef may play an important role in the innate immune response of Antheraea pernyi.