Gene Cloning,Localization And Expression Of Midgut Aminopeptidase N Isozymes From The Cabbage Looper,Trichoplusia Ni

APN plays an important role in the digestion of insect food. The digestion of protein ininsect food is a major physiological function APN has. The refraining of midgut APN activityresults in damage to larve's growth and halt of development, finally death. Due to thegrowing of transgenetic plants on large scale in recent years, insects are exposed to theenvironment with high level of Bt and are gradually becoming resistant to Bt. The resistantmechanism of insects to Bt is complex and various. One of the major mechanisms is thechange of binding spot of midgut. Cabbage looper, T.ni is one of the insects which areresistant to Bt. The isolation and identification of Cry1Ac binding protein indicate that APNis one of the receptor of Cry1Ac toxin.This thesis aimed to clarify the types of midgut APN genes of T.ni, the molecularfeatures of the subtypes, and T.ni resistant mechanism of APN to Cry1Ac. On the basis ofcreating midgut cDNA library, the study cloned APN5and APN6of T.ni by self-ligaton ofinverse PCR. In combination with obtained TnAPN1, TnAPN2, TnAPN3, and TnAPN4, allsubtypes of APN, the sequence features of the six APNs' genes were analyzed. The activityfeatures of T.ni APN and the effect of different diet on the genetic expression of T.ni APNwere revealed. The study also explored the resistant mechanism of T.ni to Cry1Ac. Thesignificance of physiochemical and molecular mechanism of resistance of T.ni APN toCry1Ac toxin in management and control of Bt resistance of T.ni. The major findings are asfollows:1. By using cDNA synthesis kits from Stratagene, the midgut cDNA library of T.ni wascreated. The original titer of the library, the combination of blue-white selection and theaverage insertion fragment were4.22×109pfu/Ml,98.8%and2.0kb respectively.2. After the removal of the signal peptide and the C-terminal prepeptide, the predictedmolecular weights of TnAPN5and TnAPN6wer112and118kDa, respectively. Twosequence features of APNs included the presence of a signal peptide at their N-termini and aprepeptide at the C-termini for the GPI anchor, the zinc binding/gluzincin motifHEX2HX18E, the gluzincin aminopeptidase motif GAMENWG and the presence ofglycosylation sites. 3. By RT-PCR, the expression of genes from the isolated six APNs in different tissueswere localized. The findings showed that the genes of TnAPN1, TnAPN3, TnAPN4, andTnAPN5were mainly in midgut, malpighian tuble,salivary gland and fat body, TnAPN6inmidgut. TnAPN2was weak in malpighian tuble,salivary gland and fat body. Enzymaticactivity assays of various larval tissues showed that aminopeptidase activities were mainlylocalized in the midgut and the specific enzyme activity per mg of midgut tissue proteins wasconstant in T. ni larvae regardless of the composition of dietary proteins and amino acids.4. By using real-time PCR, the effect of different diet on the activity expression of sixAPN subtypes T.ni larve was analyzed. The enzymatic activity assays indicated the activity ofAPN was mainly in midgut. Six APN subtypes differed in the vivo transcription level ofresistant larve. According to qRT-PCR anaysis, the transcription level of APN1gene was lowin resistant strain, and the level was0.026times as high as that of sensitive strain. Thetranscription level of APN6gene in resistant strain was39times as that of sensitive strain.There was no distinct difference between resistant strain and sensitive strain concerning thetranscription level of APN2, APN4, and TnAPN5genes.Cloning and identification aminopeptidase N gene with the digestive function helps tounderstand the relationship between the cabbage looper and BT toxin,and the role whichaminopeptidase N genes play in BT resistance.