Molecular Cloning, Analysis And Expression Of Olfactory Genes Of Heliothis Viripla Ca And Leguminivora Glycinivorella

Heliothis viriplaca (Lepidoptera:Noctuoidea) and Leguminivora glycinivorella (Lepidoptera: Olethreutidae) are important Lepidopteran pests for soybean in grow and pod period. Recently, the two pests more and more serious because of the increase area field of the free cultivate and plant soybean for several years. They are one of the main reasons of the quality and output decline. Sensillae is focus on antennae, and measure relation between the insect behavior and the environment stimulate factor of chemic and physical. It is important to insect to find host plant, food, mate and pertect prey and migrate. Therefore, the morphology and configuration is precondition of explore insect olfactory behavior and identify system. Insect olfactory identify process is very complex and many protein participate in. They are odorant binding protein, odorant receptor and odorant degrading enzymes. The proteins are important to elucidate the olfactory identify molecular mechanism. In this paper, the antenna of H viriplaca was observed by scanning electron microscopy, and use RT-PCR and RACE method to clone and analysis the olfactory related genes form antenna of H viriplaca and L glycinivorella. The Primary results are as follows:The antenna of H viriplaca was observed by scanning electron microscopy. Female and male moths are filiform and consist of the scape, pedicel and flagellum, and eight types of sensillawere found. These were sensilla trichodea, sensilla chaetica, sensilla basiconca, sensilla coelocomica, Sensilla auricillica, sensillum squamiformia, sensilla styloconica and Bohm’s bristles. The Sensillae distributed the rear and side of antenna, the scape was wrapped by squama, half of the flagellum was wrapped in order by squama, and a few of sensillum squamiformia and sensilla chaetica distributed in the squamas. Sensillae focus on flagellum, only a few Sensillae in scape and pedicel. Sensilla trichodea was the most sensillae, and sensilla chaetica was orderly on the antenna. The every little flagellum rear coverd by squamas, so most of the sensillae was in the rear and side of antenna.Five OBPs and one OR genes were cloned from the antenna of H viriplaca and L glycinivorella by RT-PCR and RACE method. They are PBP and GOBP1of H viriplaca and L glycinivorella, GOBP2and Or83b of H viriplaca, and the PBP, GOBP1and GOBP2with vector pet21b were construeted and successed expressed in E. coll induced by IPTG. The H viriplaca PBP cDNA was625bp in length and contained an open reading frame of510bp. The ORF encoded a polypeptide of170amino acid residues with a predicted molecular weight of19.0kD and a p1of5.65. A signal peptide of27residues was predicted at the N-terminus of the protein, and only one putative N-glycosylation sites was identified at the position34. The L. glycinivorella PBP cDNA was610bp in length and contained an open reading frame of504bp. The ORF encoded a polypeptide of168amino acid residues with a predicted molecular weight of19.3kD and a p1of5.55. A signal peptide of24residues was predicted at the N-terminus of the protein, and only one putative N-glycosylation sites was identified at the position52. Sequence blasted and analysed with other known insects indicated that HvirPBP and LglyPBP were characterized by six conservative Cys, which shared typical feature of OBP family. Identity of HvirPBP and LglyPBP were44%-96%and45%-70%with other known Lepidoptera insects. The kown insects had three subfamilies, and it may be connect with diversity pheromone.The H viriplaca GOBP1cDNA was973bp in length and contained an open reading frame of495bp. The ORF encoded a polypeptide of164amino acid residues with a predicted molecular weight of19.0kD and a p1of5.32. A signal peptide of19residues was predicted at the N-terminus of the protein. The L. glycinivorella GOBP1cDNA was920bp in length and contained an open reading frame of492bp. The ORF encoded a polypeptide of163amino acid residues with a predicted molecular weight of18.8kD and a p1of5.29. A signal peptide of19residues was predicted at the N-terminus of the protein. Sequence blasted and analysed with other known insects indicated that HvirGOBP1and LglyGOBP1were characterized by six conservative Cys, which shared typical feature of OBP family. Identity of HvirGOBP1and Lgly GOBP1were65%-95%and65%-82%with other known Lepidoptera insects.The H viriplaca GOBP2cDNA was624bp in length and contained an open reading frame of489bp. The ORF encoded a polypeptide of162amino acid residues with a predicted molecular weight of18.2kD and a pi of5.30. A signal peptide of21residues was predicted at the N-terminus of the protein. Sequence blasted and analysed with other known insects indicated that HvirGOBP2was characterized by six conservative Cys, which shared typical feature of OBP family. Identity of HvirGOBP2was well conserved, and between75%and99%with other known Lepidoptera insects.The H. viriplaca Or83b cDNA was1715bp in length and contained an open reading frame of1422bp. The ORF encoded a polypeptide of473amino acid residues with a predicted molecular weight of53.5kD and a p1of8.37. Two putative N-glycosylation sites were identified at the position110and170, and no signal peptide at the N-terminus of the protein. Sequence blasted found the sequence of H. viriplaca Or83b was well conserved, and over96%identity with Or83bs of other Lepidoptera moths. Analysed the sequence indicated that HvirOr83b has seven transmembrane domains, which shared typical feature of odorant receptor family.Analysed the five odorant binding proteins found they had the odorant binding protein typical feature, included small molecular weight, water-solubility acidic protein; six conservative Cys in sequences, A signal peptide was predicted at the N-terminus of the protein which secretory protein character. HvirOr83b has seven transmembrane domains, which shared typical feature of odorant receptor, and no signal peptide at the N-terminus of the protein.PBP, GOBP1, GOBP2and Or83b four genes expressed in different organs in H. viriplaca, the four genes not only expressed in the antennae of both male and female adults but also expressed in abdomen and legs. Analysed PBP in different organs by RT-PCR method found that PBP gene is main expressed in male antenna, it was30times higher than PBP gene in male antenna, and PBP gene in male antenna was3times higher than PBP other organs.