Gene Cloning, Seqencing, Prokaryotic And Tissue-Specific Expression Of CDNA Encoding Sensory Neuron Membrane Protein From Helicoverpa Assulta

The olfaction has many important effects on the behavior of insects,including the choosing of habitat,food foraging,aggregating,reproduction, signal communication,etc.In order to control the pest more specifically,it is necessary to study the mechanism of insect olfaction.The researches of insect's olfaction can explore insect's behavioral mechanism and explain the mechanism of cooperative coevolution between all kinds of behavioral responses and host plants, and provide the basis for developing effective insect attractants and repellents that have good compatibility with environment and safe to natural enemy.SNMPs (sensory neuron membrane proteins),being the only identified neuronal members of the CD36 family,belong to a larger gene family of receptor proteins characterized by the human protein CD36.SNMPs are olfactory-specific membrane-bound protein uniquely expressed in olfactory receptor neurons(ORNs),which has two-transmembrane domains with short cytosolic N- and C- terminals and a single large and central extracellular loop.Recent researches indicated that SNMPs play an important but functionally unknown role in odor detection.The insect SNMP/CD36 genes fall into three major groups,and SNMPs belong to group 3,which fall into two subgroups referred to as SNMP1 and SNMP2.This paper studied the clone and expression of Or83b gene from the antennae of H.assulta using the method of molecular biology with H.assulta as a subject.The main results are as follows:(1) The sensory neuron membrane protein(SNMP) cDNA was amplified from the total RNA of the antenna from Helicoverpa assulta by transcription polymerase chain reaction(RT-PCR) with the primer designed according to HarmSNMP (AF462067) cDNA.The results showed that the open reading-frame of SNMP was 1 572 bp,encoding 523 amino acid residues,with the predicted mature weight(MW) of 59.2 kDa and isoelectric point(IP)of 6.54.The gene was named HassSNMP,which shared high identity with sensory neuron membrane proteins from other insects.Aligned amino acid sequences of reported SNMP1 from other insects indicated the HassSNMP appearing to assemble to HarmSNMP.However,there is little difference with other SNMP1,suggesting that amino acid homology increased with the relationship of insects.Homology matrix indicated that HasssSNMP gene had the high identity with SNMP1 genes from other reported Lepidoptera insects,up to 70%.Among these genes,HassSNMP had the identity with Helicoverpa armigera SNMP1 and Heliothis virescens SNMP1 at 97%and 95%.(2) When predicting the transmembrane region for HassSNMP,we found that HassSNMP have two-transmembrane domains as HarmSNMP,locating at the same place.It showed that HassSNMP possess typical character of membrane-bound protein.(3) Two expressive vectors of HassSNMP gene were constructed successfully and the fragment containing HassSNMP gene was inserted into expressive vectors for over expression in prokaryotic cells,and induced by IPTG.The SDS-PAGE and Western bloting analysis indicated that HassSNMP gene can transcript in Escherichia coli but the protein cannot express normally.(4) The expressions of HassSNMP gene at different developmental stages and different tissues were studied by RT-PCR.Semi-quantitative RT-PCR analysis showed that HassSNMP was not only expressed in antennae,but also in head (excluding antennae),proboscis and legs of Helicoverpa assulta.However,its expression in antennae was much highest.There was no obvious diference between the expression in male antennae and that in female antennae.Moreover its expression in proboscis was much higher.HassSNMP was not expressed in thorax and abdomen. Furthermore,it was expressed in the adult,the 10th-day pupa and egg,but expressed relatively low in egg.HassSNMP was not expressed in the larva,the 1 st-day pupa,the 4th-day pupa and the 7th-day pupa.