Molecular Cloning And Quantitative Analysis And Expression Of Olfactory Genes Of Agrotis C-nigrum

Agrotis c-nigrum (Lepidoptera: Noctuoidea) is important Lepidopteran pest for Agricultureand grassland. Control methods are mainly spraying chemicals and trap adult.There is no effectiveway to Control it in root.It is one of the main reasons of the quality and output decline.It is one ofthe ways to biological control by using of insect behavior control agent.Sensilla is focus on antennae and the important part of most insect activities is the chemicalolfactory information regulation. Therefore, the morphology and configuration are preconditionwhich explore insect olfactory behavior and identify system. Insect olfactory identify process isvery complex, which many protein are participate in. They are odorant binding protein, odorantreceptor and odorant degrading enzymes. The proteins are important to elucidate the olfactoryidentify molecular mechanism. In this paper, the antenna of Agrotis c-nigrum is observed byscanning electron microscopy, and cloned and analysised the olfactory related genes form antennaof Agrotis c-nigrum by using RT-PCR and RACE method.The Primary results are as follows: The antenna Agrotis c-nigrum adults were filiform, andthey were made up of scapus, pedicle and flagella of which the latter consist of61-67segments.The outer side surface of antenna is covered with cataphylla and most of the antennal sensilla lie onit which is mainly located on the inboard antennas and windward side. There are altogether eighttypes of sensillum on the antennae: sensilla trichodea, sensilla chaetica, sensilla squamiformia,sensilla styloconica, sensilla coeloconica, sensilla papilla, sensilla auricillica and B hm bristles.The sensilla trichodea divided into two subtypes; the Sensilla auricillica is divided into twosubtypes. There are three types of sensillum on scapus and pedicle: sensilla trichodea Ⅱ, B hmbristles, sensilla squamiformia. There are seven types of sensillum on flagella: sensilla trichodeaⅠ, sensilla chaetica, sensilla squamiformia, sensilla styloconica, sensilla coeloconica, sensillapapilla, sensilla auricillica. Sensilla papilla always appears with sensilla auricillica and it is rarelydiscovered in Lepidoptera. The antennal sensilla distribution characteristics between males andfemales are basically the same, but the number of antennae length, sensilla size, there is sexualdimorphism. The female antennae11969.28μm is significantly longer than the male antennae11123.95μm. Female moths have more B hm bristles in the number of2000square micronsp=0.05level than male. The possible functions of these sensilla in the feeding process are brieflydiscussed.Three OBPs genes were cloned from the antenna of Agrotis c-nigrum by RT-PCR and RACEmethod. They are PBP, GOBP1and GOBP2of Agrotis c-nigrum and the PBP, which GOBP1andGOBP2with vector pet21b were construeted and successful expressed in E. coli induced by IPTG.The Agrotis c-nigrum PBP cDNA was1280bp in length which contained an open readingframe of516bp and ORF encoded a polypeptide of171amino acid residues with a predictionmolecular weight of18.0kD and a pI of5.34.A signal peptide of19-20residues was predicted atthe N-terminus of the protein.Only one putative N-glycosylation sites was identified. The PBPcDNA sequence has been deposited at GenBank with accession number JX978463. Sequenceblasted and analysed with other known insects showed that AcniPBP was characterized by sixconservative Cys, which shared typical feature of OBP family. Identity of AcniPBP with otherknown Lepidoptera insects was77%-88%.The Agrotis c-nigrum GOBP1cDNA was1259bp in length, which embodyed an open readingframe of486bp and ORF encoded a polypeptide of161amino acid residues with a presagemolecular weight of18.6kD and a pI of5.14. A signal peptide of16-17residues was presaged atthe N-terminus of the protein. The PBP cDNA sequence has been deposited at GenBank withaccession number JX978464. Sequence blasted and analysed with other known insects showed that AcniGOBP1was characterized by six conservative Cys, which shared typical feature of OBPfamily. Identity of AcniGOBP1with other known Lepidoptera insects was71%-97%.The Agrotis c-nigrum GOBP2cDNA was702bp in length, which included an open readingframe of489bp and ORF encoded a polypeptide of162amino acid residues with an indication of18.1kD of molecular weight and a pI of5.20. A signal peptide of21-22residues was indicated atthe N-terminus of the protein. The PBP cDNA sequence has been deposited at GenBank withaccession number JX978465. Sequence blasted and analysed with other known insects indicatedthat AcniGOBP2was characterized by six conservative Cys, which shared typical feature of OBPfamily. Identity of AcniGOBP2was well conserved, and between84%and99%with other knownLepidoptera insects.It found from three odorant binding proteins they had the odorant binding protein typicalfeature, included small molecular weight, water-solubility acidic protein and six conservative Cysin sequences. A signal peptide was predicted at the N-terminus of the protein which has secretoryprotein character.