Gene Cloning,Expression And Functional Analysis Of Aminopeptidase N3(GmolaAPN3)from Midgut Of The Oriental Fruit Moth, Grapholitha Molesta(Busk)(Lepidoptera: Olethreutidae)

The oriental fruit moth?Grapholitha molesta?,is one of the most serious pests that attack pomefruits all over the world.The larvae have a typical harmful habit of bore through shoots and fruit of rosaceae.Over-reliance on chemical pesticides is not an ideal way to prevent and control Grapholitha molesta.The natural enemies is killed,the environment is polluted,lead to pesticide residues and lead to insect resistance by Overuse of chemical pesticides.Bt insecticidal protein?ICPs?is highly specific to target insects and is harmless to humans and livestock.In addition,using Bt to control the Grapholitha molesta is efficient,non-polluting and no hurting to beneficial insects,and full compliance with environmental protection.The Bt toxin bind receptors on the surface of BBMV?Brush border membrane vesicles?in the midgut of insect,resulting in the death of insect.Many Bt receptors in the midgut of Lepidopterous insects have been discovered and studied in depth.However,no similar studies have been reported for Grapholitha molesta.This research uses selected Grapholitha molesta sensitive to Bt toxin as the object of study.The gene of GmolAPN3 was cloned,and the expressed by using molecular biology techniques.And the expression levels of this gene in different development stages and different tissues of the larvae were analyzed by quantitative real-time PCR.In addition,the toxicity of Cry1Ac toxin at different concentrations against 2nd instar larvae,and the expression changes of GmolAPN3 gene in larvae were studied after 2nd instar larvae were fed on Cry1Ac toxin.Finally,interfered GmolAPN3 gene by RNAi technology and measured the mortality after fed Cry1Ac toxin.The results will provide important guidance for definite the mechanism of Bt,development the preparation of Bt which is prevent Grapholitha molesta efficiently and transgenic Bt crops.The results are summarized as follows:1.The gene GmolAPN3-encoding cDNA sequence was cloned from midgut of Grapholitha molesta by degenerate PCR and RACE techniques.The results indicated that the full-length of the GmolAPN3 coding gene is 3 249 bp.GmolAPN3 contains an open reading frame of 3 021 bp that encodes for a 1 006 amino acid residue protein with a predicted mass of approximately 113.9 kDa and an isoelectric point of 4.72.It has typical structures that APN of other lepidoptera insect have.The putative protein sequence includes nine N-linked and seven O-linked glycosylation sites,a highly conserved GAMEN motif that forms part of the active site,and a zinc metal binding site motif of HEXXH?X?18E that is typical of the active sites of zinc-dependent metalloproteases.In addition,it contains a cleavable N-terminal signal peptide with 19 amino acids,as well as a glycosylphosphatidylinositol?GPI?anchor signal peptide with 17 amino acids at the C-terminus.It has GluZincin superfamily Aminopeptidase N domain and ERAP1-like C-terminal domain which are also the typical characteristics of lepidopteran GmolAPN3 proteins.The analysis of phylogenetic tree showed that GmolAPN3and APN3 of other lepidopteran insect in the same branch,as class3 member in the family of APN.GmolAPN3 have close relationship with Sesamia inferens,Plutella xylostella,Helicoverpa armigera and Heliothis viescens.2.The GmolAPN3 gene was divided into four segments for the prokaryotic expression and to induce the expression which obtained the best condition.3.Developmental expression profiles revealed that the GmolAPN3 gene had expression level in larva than in other development stages,with the highest expression level in the 2nd instar larva,and the lowest expression in the egg and pupa.Tissues expression profiles revealed that the GmolAPN3 gene was mainly expressed in the midgut.4.After feeding Cry1Ac toxin to 2nd instar larvae of Grapholitha molesta,the toxicity regression equation of Cry1Ac toxin was:y=-1.046+1.014x,LC₅₀=10.768?g/g.5.The expression of the GmolAPN3 gene in the 2nd instar larvae which fed on Cry1Ac toxin was differently.When fed with low concentration?1.592?g/g,3.273?g/g?toxin,the expression level of GmolAPN3 gene was lower than CK,and showed a tendency of decreasing first and then increased with a low mortality rate.When fed with high concentration?10.768?g/g,35.427?g/g?toxin,the gene expression showed a decreasing trend after a transient increase,the mortality rate a high.6.The interference efficiency of GmolAPN3 gene after fed 2nd instar larva with 7?g/g dsAPN3 reached 60%.The mortality rate of 2nd instar larvae of Grapholitha molesta after fed with Cry1Ac toxin was decreased dramatically.A preliminary deduction is that GmolAPN3 has the receptor function of binding proteins of Cry1Ac toxin in Grapholitha molesta.