The Genomic Identification And Functional Study Of A Sensory Neuron Membrane Protein (SNMP) Gene Family In The Silkworm, Bombyx Mori

Olfaction plays a vital role for insects in search of food, mating and oviposition sites. Presently great progresses have been achieved on the olfactory mechanism. Depending on olfaction system insect could recognize the volatile odorants in the outer enviroment, and with the interaction of various proteins the signal was transducted to central neuron system. The sequencing of insect genome privided an excellent opportunity of identification of funcional proteins and candidates involved in olfaction. Sensory neuron membrane protein (SNMP) belongs to CD36 superfamily, which has 2 transmembrane domains at N-terminal and C-teiminal.The first SNMP was identified from the antennae of Antheraea polyphemus.After that several SNMP ortholog have been found in other species.There are reports on Drosophila which suggest sex pheromone cVA could bind and active LUSH protein, then the complex was transfered to Or83b/Or67d dimer receptor site with the help of SNMP1(CG7000), and finally triggered the downstream pathway. The SNMP1 from silkworm shared high identity with Drosophila SNMP1,so we speculate that they have conserved function.Bombyx mori is the model insect of lepidopteran, and also the economically important agricultural insect. Recently completed fine map of silkworm genome provide a basic platform of identification of important genes and functional study. Many gene families, such as Chemoreceptor family and Odorant binding protein family, have been identified. In the current study, we have identified a SNMP gene family, characterized the sequence feature and analyzed the phylogenetic properties among different insect species. Meanwhile, in situ hybridization and RNA interference were performed to investigate the function of SNMP1 gene. The major achievements are as follows: 1.Identification and bioinformatics analysis of SNMP gene family from silkworm genome15 members of SNMP gene family were identified from silkworm genome, including 2 previous identified. These candidates encode 400 to 600 amino acids with conserved intron/exon splice position. They are located on 5 chromosomes, some of which clustered from 2 to 4 genes. Most genes within the cluster are in the same orientation, which indicates they may arise from gene duplication events. Phylogenetic analysis of this gene family from 5 species showed that the SNMP gene family could be categorized as 3 groups, and most silkworm SNMPs have orthologs with Diptera. Recently CG7000 in Drosophila has been proved playing a role in detection of sex pheromone. The high identity between SNMP1 and CG7000 suggests SNMP1 might function similarly in the silkworm. Notably, there is a silkworm-specific lineage, SNMP11-15, which would arise by a gene expansion within this species or lepidopteran.2.Spatio-temporal expression pattern of the SNMP gene family in silkwormThe results of RT-PCR showed that the SNMPs are widespread expressed in different tissues, and most of them are continuously expressed during the development. SNMP1 was expressed exclusively in the male antennae suggesting it may be involved in the olfaction. SNMP2 was firstly reported as the homolog of SNMP1 and expressed in almost all the detected tissues which might provide the evidence for quite different function. SNMP10 was highly expressed in midgut. The expression level of SNMP10, as well as testis-specific SNMP11 and widely expressed SNMP14 could be regulated after the invasion of microorganism according to the microarray data. Because members of this family were identified as the scavenger receptor B, we speculate these genes are likely to participate in the immunoreactions.3.SNMP1 could be involved in the courtship of silkwormThe location of SNMP1 gene was confirmed by in situ hybridization using DIG labelled cRNA probe. The result shows that SNMP1 expressed in the olfactory sensory neurons in the antennae. RNAi was performed by injection of synthesized dsRNA into the male pupae. The results of Real time PCR indicated that the SNMP1 was successfully suppressed. We designed behavior assay to analyse the impact of RNAi, and found that the RNAi male moths spent longer time than control finding females and starting copulation. Moreover, some even failed to seek mate. Combined the above all results, we put forward the hypothesis that SNMP1 is involved in the courtship and plays key role in the recognition of sex pheromone.