Genomic Structure, Expression And Function Analysis Of The Odorant Receptor Family In The Silkworm, Bombyx Mori

Insects composed the most vairous species in the living world, they existed in divergent niches.Some insects took advantages for human being, but many insects are pests, they destroied millions of crops and forests, even threterned the public health. Insects have evolved robust and extremely sensitive chemosensery systems that are elicited by chemical cues in their surroundings, then innervated the behaviers such as searching for food, avoiding predators, identifing mates and detect oviposition places. Chemosensery systems can be devided into two parts by function, olfactory and gustatory. Olfactory system is responsable for detecting volatile chemical cues, while gustatory system is responsable for detecting soluble chemicals.Elucidation the molecular mechanisim of insect is good for controlling the popelation of pests, reducing the damage of argreculture products and keeping public health.Bombyx mori belonged to the complete metamorphosis insects, it has four development stages, eggs, larval, pupae and adult. Only in the larval stage can the silkworm ingest mulberry leaves and storage energy for all the other stages;aldult stage is responsable for searching for mates and laing eggs.However, the Bombyx mori can not move in egg or pupae stage. Such distinct fuctions in different development stages leads it to an excellent model for researching the molecular mechanisim of insect olfactory system.Even more, the silkworm is considered to be the model of Lepidoptera, elucisating the mechanisim of silkworm olfactory system is good for silk indestry, but also give some cues for Lepidoptera pests control.Based on the new assembled silkworm genome sequence, we have take a primary reasearch for the bombyx mori olfactory receptors which have been reported, the main results were as follows,1.Genomic analysis of the B.mori olfactory receptorsThere are 66 olfactory receptors in the genome of B.mori have been reported hitherto.They are mapped to 19 of 28 chromesomes in the B.mori.48 ORs genes are clustered in their locis of chromosome, and arranged in tandem array manner. The most scafold have clustered six ORs genes, there are 2 ORs genes clustered at the least case. However,the left 18 ORs genes are single loci in the chromesome.Multiple alligments blast by ClustalX, the results display that all the 66 ORs genes are clestered into 8 clanes,the similar percentage of their deduced amino acids are very low. They encoded 117 to 472 amino acids residues in length,and belonged to the seven-transmembrane receptor protein superfamilies.Exon/intron analysis results show the number of their introns are arranged from 0 to 9.The most introns is BmOr2, three genes are single exons, BmOrl3,BmOr38 and BmOr49, respectively.2.Expression characteristion of BmOrs on transcriptional levelThe ORs genes of B.mori are broadly expressed in various tissues and development stages by RT-PCR, such as larval antenna, cuticles and maxillary plaps, adult antenna, heads,legs, wings and caudal sheath. Many ORs genes have sex dimorphism in the transcriptional level.We found 7 male-biased OR genes,they are BmOr10,BmOrll,BmOr21,BmOr33,BmOr44,BmOr59 and BmOr-63.Alternatively, there are 7 female-biased OR genes,BmOr20,BmOr24,BmOr37,BmOr41,BmOr48,BmOr49,BmOr58, respectively. A large number of genes expressed in legs, wings and caudal sheath are male-biased.3.Reduced expression the BmOr2 leads to olfactory sensitivity damaged in the B.moriWe performed RNAi stratage to research for the primary function of BmOr2. DsRNA of BmOr2 were manually synthesised by kit, then injected in to the pupae stage 4-5th day of B.mori. When examined the aldult sensitivity to several volatile chemicals including Benzaldehyde,2-Phenylethanol, cis-3-Hexenol and Terpineol, we found moth had a reduced frequency of electrophysiology response to these compounds.Moreover, male moth had a delayed timescale in the behavior of searching for their mates.We concluded that BmOr2 had certain impotant role in the odor sensitivity in the B.mori.4. Transgenic silkworm expressing hairpin Bm0r2 RNAi lineIn order to producing stable genetic line of Bm0r2 RNAi individuals, we constructed transgenic RNAi vector, then introduce it into the eggs of silkworm by microinjection.We screened 11 brooms in their offfsprings,which showed green flroursence positive.We detected 3 of 11 individuals by Southern bloting, the results showed there were two brooms were single copy, while another was double copies.