Studies On The Odorant Binding Protein Genes Of Silkworm, Bombyx Mori L.

Domestic silkworm Bombyx mori is the closest insect with the production and living of homo-sapiens. Bombyx mori is the only Lepidopteran insect whole genome sequence available. And it has been considered as the lepidopteran model insect by The International Lepidoptera Society, TILS. In the past decades years, silk moths were also as the model insect utilized in moth olfactory reception conduction research, the odorant binding proteins (OBPs) were on the focus, especially the pheromone-binding proteins (PBPs). OBPs in insects play a primordial role in foraging, courtshiping, reproduction, and chemical communication with the environments. Accordingly, researches on the OBPs of silkworm would further recognize the functions, and illuminate the mechanism of silkworm olfactory, and be utilized in pests governing and in beneficial insects rearing. For those reasons, we investigated the genetic structure, chromosome location, expression profiles, gene function of OBPs, and the molecular markers related to feeding habit to artificial diet of silkworm utilized bioinformatics, gene localization, expression profile analysis, gene cloning, RNA interference (RNAi), molecular evolution analysis, and molecular markers analysis. The results as follows:1 We analyzed the OBP family members of Bombyx mori released in NCBI with bioinformatics methods and softwares, and found the family members located on the 7 disparity chromosomes, and emerged 4 gene clusters. The genetic structure showed obviously polymorphism. The disparities were fairly larger of nucleotide sequence similarity and genetic distance. The electronic expression profiles were extensive, and many genes expressed in different tissues. The family had similar biochemistry character to insect OBPs, and two were transmembrane proteins, 5 embrane-associated proteins. The amino acids had low level sequence identities and remote genetic distances, but cysteine and tryptophan were very conservative. There possessed Minus-C group like Drosophila melanogaster and other insects, and no Plus-C group in the family. The results indicated OBP probably functioned in many aspects in silkworm.2 Pheromone binding proteins/general binding proteins(PBPs/GOBPs) were an important monophyletic group of Lepidoptera. We mapped the genes in the genome of silkworm. The results exhibited those genes organized in the nscaf3052 of Chromosome 19^th in form of gene cluster, with similar gene structure and identical transcript direction, indicated them originated from homologous gene duplication and possessed functional similarity probably. Analysis the expression profiles in different tissues in different female and male tissues during pupal and adult stages, the results revealed that those genes possessed conspicuous temporal and special differences, the maximum relative expression level occured in the antennae, and lower in other most no-olfactory organization without significant sex differences. The results indicated the gene cluster may function as a olfactory organism, and others undiscovered. Otherwise, taxis experiment of silkworm adult males showed olfactory reaction between adults was the responses to sexual attraction and independence to attractant or repellent, the major function of OBPs were related to receipt sexual pheromone irritation.3 We analyzed the predicted tertiary structures and functional sites, gene expression profiles of the whole stages, and function of ABP and ABPX by RNAi in the 5th larva stage. The two proteins were significantly differences, and belonged to different groups. They were transcribed highly in olfactory tissues (organism), and by no means merely in those tissues, but expressed in all most tissues detected. Removal olfactory organisms experiment showed the two genes possibly correlated to the taxis to ingestas in larva stage. But the transcript levels of targeted genes significantly decreased after injected with dsRNA or oral application of bacterial expressed dsRNA. RNAi did not cause olfactory malfunction of silkworm, but postponed the developmental process of the 5th larva significantly. Those results indicated the two genes probably concerned with the taxis to ingestas and shouldered other important physiological functions related to development in larva stage.4 Sex pheromone signal transduction in moths was focused on PBP subfamily member and OR (olfactory receptor) genes at present. It formed a paradigm for the study of insect chemical communication. PBPs, OR1 and OR3 played an important role in the process of the male silkmoths perception and location of the females. For the purpose of finding out the molecular mechanism of difficult hybridization,and understanding the evolution of related genes to sexual recognition between the demostic and wild silkworm. We cloned the 3 PBP and 2 OR genes from wild silkmoths Bombyx mandarina. The results, of sequence analysis of coding regions, revealed that the genetic distances were very intimate and the base transversion was the predominant form of basic mutations. Secondary structure predicted suggested mutation sites had no influence on the adjoining regions, and nor on the functional sites. They are presumably no functional differences in the two related moths,So the two related species can receipt, recognize each other, and it founded the theoretical evidence for the exploitation of wild silkworm through mutual cross. Meanwhile, it was the molecular evidences of domestic silkworm originated from wild silkworm. 5 General OBPs (GOBPs) were considered as the binding protein of variety of general odorant molecules associated with food and the environment. The molecular evolution based on GOBP subfamily sequences of 13 insects released by NCBI indicated there presented high identity in the GOBP1 and GOBP2 amino acid and nucleotide sequences, and low level sequence divergence. The genetic relationship was very close between the two genes. Also, cysteine and tryptophan were the most conservative amino acids. But the two genes originated from two different ancestral gene, and had been duplicated prior to the divergence of those 13 species.Theω(ka/ks) analysis based on the nucleotide and amino sequences indicated Ka/Ks ratios were generally small. Only one positive selection was detected during the evolutionary process of GOBP1 between Helicoverpa assulta and Helicoverpa armigera. Those results demonstrated GOBPs were probably functional similarity among insects.6 Correlated characters of feeding habit on artificial diet of silkworm perhaps regulated by no-feeding genes according to the molecular markers experiments. Polymorphism of RAPD (Random Amplified Polymorphic DNA) markers exceeded those of SSR (Simple Sequence Repeat). And special marks of SSR mainly came from the chromosome 10th, 11th, 17th,19th, 20th, 27th, and 28th. The major were on the chromosome 20th, and many special bands amplified from the parents with high feeding ability. It indicated the genes related foraging artificial diet located on the chromosome probably. And special bands amplified by the SSR marks of chromosome 17th mainly came from the parents with low feeding ability. The no-feeding genes perhaps located on it. Those results founded the base of determination molecular markers of feeding habit related characters and assisting molecular seed breeding. It would promote sericulture,and benefit in pests control and useful insects breeding.