Cloning,Expression And Functional Analysis Of Sensory Neuron Membrane Proteins (SNMPs) From Delia Antiqua Megien (Diptera:Anthomyiidae)

Delia antiqua Meigen(Diptera: Anthomyiidae)is one of the most serious subterranean pest of Allium plants in China as well as many parts of the northern hemisphere.To date,the main control method against Delia antiqua is chemical control.Long-term use of chemical agents is not only easy for developing insecticide resistance,but also causing environmental pollution.Insects have complex olfactory systems that can perceived variety of pheromones and volatiles.Synthetic sexual attractants and plant volatiles were used for monitoring and control insect pest,has obtained the remarkable effect.Sensory neuron membrane proteins(SNMPs)are critical olfactory proteins in insects and act as markers for pheromone detection.Therefore,the study of the function of SNMPs will be benefit for developing pheromone-mediated pest management methods.In this study,we have cloned the open reading frame of DantSNMP1 and constructed phylogeny analyses.In addition,we also studied the expression patterns of DantSNMP1 and DantSNMP2 in different tissues,and carried out immunofluorescence localization of DantSNMP1 distribution in the antennae.The results are as follows:1.The open reading frame of DantSNMP1 was cloned and identified using a combination of homology-based PCR cloning techniques and rapid-amplification of cDNA ends(RACE)method.The gene was named DantSNMP1 and submitted to GenBank for accession number MK541687.The open reading frame length is 1608 bp,which encodes 535 amino with a molecular weight of 60.2 KD and an isoelectric point of 5.39.Transmembrane regionanalysis showed that DantSNMP1 had the typical characteristics of CD36 protein family which have two conserved transmembrane domains and a large extracellular loop consisting of six conserved cysteine residues.Phylogenetic analysis showed that DantSNMP1 belonged to the SNMP1 subtype and had high similarity with the amino acid sequence of SNMP1 of Dipteran insects(65%-78%).2.The tissue expression patterns of DantSNMP1 and DantSNMP2 were analysed by real-time quantitative PCR.The results revealed that DantSNMP1 was antenna-specific and the expression of DantSNMP1 in male antennae was significantly higher than that in female antennae,suggesting that DantSNMP1 has olfactory function.However,DantSNMP2 was expressed in antennae,foot and wing,among which the expression in male antennae was significantly higher than that in female,and the expression in foot and wing was similar between sexes,suggesting that DantSNMP2 has olfactory and gustatory functions.3.The constructed prokaryotic expression vector DantSNMP1-pET-28a(+)was introduced into E.coli Rosetta cells,and 44 KD recombinant protein in the form of inclusion bodies was induced.DantSNMP1 recombinant protein was used to induce polyclonal antibody by immunizing New Zealand white rabbits,and the immune activity polyclonal antibody(titer: 1000 x 2 ^ 10)was obtained.4.The obtained polyclonal antibodies were used for immunohistochemistry analysis,and the results showed that DantSNMP1 was expressed in the sensilla trichodea which were sensitive to sex pheromones,suggesting that DantSNMP1 has the function of detecting sex pheromones.