Expression Pattern And Prokaryotic Expression Of Pheromone Binding Protein 2 And Glutathione S-transferase Gene From Helicoverpa Assulta (Guenée)

The sensitivity and specificity of identifying volatile compounds play an important role in existence and reproduction in insects. These interactions consist of sequential steps including odorant binding, reception, transformation and termination of the signal from chemical message into an electrical message and etc, in which many kinds of proteins work. Pheromone binding proteins (PBPs), identifing the pheromones possessing similar structure, and glutathione S-transferases (GSTs), contributing to signal termination of aldehyde, especially sex-pheromone odorants, play an important role in communication between male and female insects. In order to provide the basis for the further analysis of communication mechanism between male and female insects, the cloning, expression pattern and prokaryotic expression of PBPs and GSTs gene of Helicoverpa assulta were conducted. The main results are as follows:(1)The open reading frame and 3′end full-length cDNA of a novel pheromone binding protein 2 gene was cloned from Helicoverpa assulta(Guenée)antenna by using RT-PCR and RACE methods, which was named HassPBP2 ( GenBank accession No. EU316186). The cloning and sequencing results showed that the full length of open reading frame in HassPBP2 was 450 bp, encoding 149 amino acid residues with the predicted molecular weight and isoelectric point of 16.9 kD and 5.56, respectively. Gene structure analysis revealed that HassPBP2 consists of 3 exons and 2 introns with length of 90 bp and 261 bp, respectively. Amino acid sequence analysis showed that the deduced amino acid sequence of HassPBP2 had the characteristics of odorant binding proteins. HassPBP2 had 34%~91% identity with PBPs of other Lepidoptera moths, and shared 91% identity with the PBP2 sequences from Helicoverpa armigera and Heliothis virescens.(2)The expression pattern of HassPBP2 was studied by using intron-spanning primers and semi-quantitative RT-PCR, the result revealed that HassPBP2 transcript was observed clearly from mid-stage pupa to mid-stage adult, but not in the egg, larva and early-stage pupa, and only expressed in the antennae of both male and female adults.(3)The expressive vector pGEX-HassPBP2 of HassPBP2 was constructed successfully and the fragment containing HassPBP2 was inserted into the expressive vector for over expression in prokaryotic cells, and induced by IPTG. The SDS-PAGE analysis indicated that the fusion protein was expressive successfully.(4)The open reading frame of a novel glutathione S-transferase gene was cloned from the antenna of male moth by using RT-PCR and RACE methods (GenBank accession No. EU316186). The cloning and sequencing results showed that the full length of open reading frame in the glutathione S-transferase gene is 654 bp, encoding 217 amino acid residues, and the predicted molecular weight and isoelectric point are 24.8 kD and 7.76, respectively. Gene structure analysis revealed that the glutathione S-transferase gene consists of 6 exons and 5 introns with length of 415bp,513bp,296bp,333bp和269bp. Based on the sequence similarities and phylogenetic tree,the sequence obtained belongs to the epsilon family,a kind of insect-specific protein, named as HaGSTe1.(5)The expression pattern of HaGSTe1 was described by using intron-spanning primers and semi-quantitative RT-PCR, the results showed that HaGSTe1 transcript was clearly observed in middle-stag larvae, late-stage larvae, late-stage pupae and late-stage adult, but not in the eggs, early-stage larvae and early-stage to middle-stage pupae.In adult, it was only observed in the antennae of both male and female and proboscises. The expressive vector pGEX-HassGSTe1 was constructed successfully.(6)The open reading frame cDNA of another novel glutathione S-transferase was cloned from the abdomen of moth by using RT-PCR method(GenBank accession No. GQ856239). The results showed that the full length of open reading frame in this gene is 636 bp, encoding 211 amino acid residues, and the predicted molecular weight and isoelectric point are 24.2kD and 6.66, respectively. Based on the sequence similarities and phylogenetic tree,the sequence obtained belongs to the epsilon family,named as HaGSTe2.( 7 ) The expression pattern of HaGSTe2 was described by using semi-quantitative RT-PCR and revealed that HaGSTe2 transcript was clearly observed from the antennae of both male and female, proboscises, heads without antennae and proboscises, thorax, abdomen, legs and wings, and total developmental stages, including egg, larvae, pupae and adult.