Gene Cloning And Expression Analysis Of Odorant Receptor And G Protein α Subunit From Helicoverpa Assulta (guenée)

Olfaction plays a crucial role in insect survival and reproduction. Insect odorant perception is a complex process. The initial steps in odor detection involve the binding of an odor to the odorant receptor (OR) displayed on dendrites of olfactory sensory neurons (OSNs). There is a widespread assumption that insect ORs belong to the family of G protein–coupled receptor (GPCR). However, there are several challenging evidences against the assumption. In this research, the genes encoding OR and G proteinαsubunit from Helicoverpa assulta, and the relationship between the two proteins were studied. The main results are as follows:(1) The full length cDNA encoding Or83b-like receptor of Helicoverpa assulta was isolated by using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Nucleotide sequence analysis revealed that the cDNA contains a putative coding region of 1422 bp, 5′untranslated region (UTR) of 135bp and 3′UTR of 389bp. Sequencing analysis showed that the open reading frame (ORF) of the cDNA encodes a protein of 473 amino acids with a predicted molecular weights (MW) of 53.51kD and isoelectric point (PI) of 8.73. The predicted protein obtained, containing seven putative transmembrane domains, shares high amino acid identity with other Or83b family receptors. The results suggest that the gene obtained is one member of Or83b family genes and this gene was hence named as HassOr83b. This sequence has been deposited in the GenBank database with accession number EU057178.(2) A fragment of HassOr83b was amplified from genomic DNA. The sequencing of the DNA fragment generated a total of 1202bp nucleotide sequence. The intron position for the fragment was determined on the basis of a comparison of cDNA with the genomic sequence. The intron sequence in the fragment is 1087bp in length. The partial sequence of HassOr83b from genomic DNA has been deposited in the GenBank database with accession number EU497670.(3) The expression of HassOr83b transcript in several adult tissues as well as during preadult stages was described by using intron-spanning primers and RT-PCR. The results showed that the HassOr83b transcript is clearly observed in the antennae, labial palps and proboscises, but not in bodies, wings and legs. In addition, HassOr83b is also expressed in several preadult stages, including early-stage larvae, late-stage larvae and pupae, but not in embryos.(4) To assess HassOr83b are in fact expressed in antennal sensory cells, in situ hybridization experiments were performed. In situ hybridization of antennal sections with the HassOr83b antisense RNA probe provided clusters of labelled cells, most likely representing sensory cells housed in trichodic sensilla.(5) A 1103bp cDNA encoding Gαq protein of Helicoverpa assulta was isolated by RT-PCR. Nucleotide sequence analysis revealed that the cDNA contains a putative coding region of 1062 bp, 5′UTR of 21bp and 3′UTR of 20bp. Sequencing analysis showed that the ORF of the cDNA encodes a protein of 353 amino acids with a predicted MW of 41.5kD and PI of 5.15. The predicted protein obtained shares high amino acid identity with other known Gαq subunits. This sequence has been deposited in the GenBank database with accession number EU057176 and this gene was named as HassGαq.(6) The expression pattern of the HassGαq gene transcript was examined by RT-PCR analysis of RNA isolated from several developmental stages and adult tissues of Helicoverpa assulta. The HassGαq transcript is expressed in all adult tissues tested, as well as throughout preadult development. The expressions of HassGαq are not tissue specific.(7) HassGαq gene was constructed into expression vector pGEX-4T-2 for over expression in prokaryotic cells. The SDS-PAGE and Western blot analysis showed that induced by Isopropyl-β-D-Thiogalactoside (IPTG), the GST-HassGαq fusion protein is expressed in Escherichia coli BL21, and its MW was found to be about 66kD nearly equal to the predicted.(8) The specific band of expression was excised from the gel and used to immunize the mice. The antiserum was collected from the immunized mice. Male antennal homogenate of H. assulta was separated by SDS-PAGE and detected by Western blot using the antiserum prepared, and anti-Gq/11αantiserum purchased respectively. Of the tested sera, anti-Gq/11αantiserum detected a single immunoreactive band with an apparent molecular mass of 40 kD that was consistent with the molecular weight of HassGαq protein, and the antiserum prepared in this research detected several bands. The results indicated that anti-Gq/11αantiserum can recognize HassGαq protein with high specifity.(9) The expression sites of HassGαq in adult male antennae were examined by immunocytochemistry method using anti-Gq/11αantiserum against HassGαq. The results revealed that HassGαq was associated with trichodic sensilla and basiconic sensilla as immunolabeling were typically observed inside and at the base of the two kinds of sensilla.(10) The location of HassOr83b and HassGαq in the male adult antennae of H. assulta supports the view that HassOr83b belongs to the family of GPCR.There is no publication concerned with studies above.