Gene Cloning And Expression Analysis Of Sensory Neuron Membrane Protein Gene From Apis Cerana Cerana (Hymenoptera: Apidae)

Insect sensory neuron membrane protein (SNMP) is located in the sensory neurone, playing animportant role in the formation of insect olfactory. In order to explore the molecular function of SNMPin the formation of olfactory, we take chinese honeybee (Apis cerana cerana) as the research object, thefull length of sensory neuron membrane protein gene was obtained by RACE method in the antennae ofworker bees. We also did phylogenetic analys is of SNMP gene, and studied the expression in thedifferent tissues and the immunolocalization in antennae of A. cerana cerana.The main results are as follows:1. We got the full length cDNA of SNMP by RACE method, and designed a pair of specificprimers based on the open reading frame (ORF), cloned sensory neuron membrane protein gene codingregion by using RT-PCR method. Sequence analysis results showed that the ORF is1563bp in length,encoding520amino acids with the predicted molecular weight of58.02kD and the theoreticalisoelectric point of5.83. Tmap software was used to predict its transmembrane region, the resultshowed that the protein was double-transmembrane protein, with the typical characteristics ofmembrane protein. This protein named as AccSNMP1with GenBank accession no. KC012595.2. Multiple sequence alignment indicated that AccSNMP1from A. cerana cerana shares differentidentities with those from other nine insects at the amino acid level. The AccSNMP1gene from A.cerana cerana has high amino ac id sequence identity with that of Apis mellifera and Bombus impatiens(above90%), while Lepidoptera and Diptera insects SNMP sequence had multiple different amino acids,with a identities of50%-65%at the amino acid level, and has the lowest amino acid sequence identitywith that of Tribolium castaneum (22.7%). We selected the SNMP sequences in a variety of insects toconstruct a phylogenetic tree, The phylogenetic analysis indicated that A. cerana cerana species has aclose relationship with A. mellifera and B. impatiens, and has a far relationship with oher insects. Itshowed that the gene is relatively conservative and has the same basic function, there is also a certainamount of variation, indicating that the function of the protein may be different among different species.3. Tissue expression profiling of AccSNMP1quantified by real-time PCR demonstrated that it wasexpressed in most of the tissues in worker bees, expecially highly expressed in antennae and legs of A.cerana cerana, showing a significant difference with that in thorax, abdomen, proboscis and headwithout antennae and proboscis (P<0.05). Organizations in expression levels from high to low are asfollow: antennal> legs> thorax> proboscis> head (without antennae and proboscis)> abdomen.4. A recombined plasmid pEASY-E1-AccSNMP1was constructed using pEASY-E1as the fusedexpression vector, and AccSNMP1was expressed successfully after induced with IPTG in BL21(DE3)strain of Escherichia coli. We got the expected molecular weight protein bands after SDS-PAGEelectrophoresis, and then the protein was identified by mass spectrometry and Western blot analysis, theresults showed that the protein is recombinant proteins expressed in E. coli of A. cerana ceranaAccSNMP1gene. 5. Immunohistochemical localization study of AccSNMP1had been done in A. cerana cerana. Weprepared polyclonal antibody in the use of AccSNMP1prokaryotic expression product. Taking thepolyclonal antibody as the first antibody, Alexa Fluor488-labeled goat anti-rabbit IgG (H+L) antibodyas the second antibody, we did the immunohistochemical analysis. A large number of expression ofAccSNMP1was found in the sensilla trichodea of antennae. It showed the AccSNMP1may be theimportant function factor in olfactory recognition in antennal of A. cerana cerana.