Cloning And Function Analysis Of General Odorant Receptor Genes From Cotton Bollworm,Helicoverpa Armigera

The olfactory system is a key physiological element which insect dependent on it in the process of survival and reproduction among other animals in the environment. Olfaction plays an indispensable role in manage insect behavior such as appropriate mating partners choice, food selection, host select and so on. Helicoverpa armigera is one of the important drill decayed pests in cotton boll period, main bored bud, flower and leaves. With the continuous improvement of various crops cropping index, the incidence of H. armigera provided a rich diets and favorable breeding sites, coupled with enhanced insect resistance, the harm of lead to H. armigera has a tendency to increase year by year. Therefore, looking for new technology to become the primary problem of prevention and control H. Armigera, many scholars have made the odorant receptors as a research focus. We cloned full length sequences of two odorant receptor genes by PCR and RACE techniques. The structure prediction, sequence alignment and phylogenetic analysis of these two odorant receptors were performed using different bioinformatics softwares. The expression profiles of these genes in different tissues were investigated by using semi-quantitative RT-PCR. The functions of three ORs genes were studies by using two-electrode voltage clamp and electroantennogram response. The main results are as follows:1^Three full-length cDNA sequences encoding odorant receptors were obtained and named as HarmOR9HarmOR10and HarmOR29. Sequence analysis showed the full-length of HarmOR9gene was1206bp, which encoded a polypeptide of401amino acids. The full-length of HarmOR10gene was1173bp, which encoded a polypeptide of390amino acids. The full-length of HarmOR29gene was1188bp, which encoded a polypeptide of395amino acids.Sequence alignment and phylogenetic tree analysis of these three odorant receptors with reported odorant receptors from Lepidopteran insects, including Helicoverpa assulta, Bombyx mori, Heliothis virescens and H. armigera showed that these three odorant receptor genes were obviously different from pheromone receptors, but clustered together with other general odorant receptors. The results of semi-quantitative PCR showed that these three odorant receptor genes were highly expressed in antennae. HarmOR10biased expressed in male antennae than in female, HarmOR9and HarmOR29there were no difference between male and female. HarmOR9were micro-expressed in male antennae and not expressed in others tissues; HarmOR10were highly expressed in female genitals and not expressed in others tissues; HarmOR29were not expressed in others tissues.2^The Xenopus-based functional study indicated that benzaldehyde as HarmOR10receptor specificity ligands. The concentration gradient records indicated HarmOR10is most intense when benzaldehyde concentration is0.001mol/L and reaches smell saturated. HarmOR9and HarmOR29found no ligands.3、The electroantennography reaction indicated that H. Armigera adult has response to these17kinds of volatile odors. Male and female worms in the same region as the research object, H. Armigera in Beijing by most odors stimulation showed male response is stronger than female and few odors (such as phenylacetaldehyde, Myristic acid, Ethyl palmitate, Methyl benzoate, etc.) have opposite response; the study of H. Armigera in Langfang showed male response is stronger than female to all the odors and no obvious difference. Different regions of the same sex of H. armigera as the object of study, the results showed that the response of H.armigera in Langfang is obvious than H. armigera in Beijing and males have more sensitive reaction. The semi-quantitative PCR result of HarmOR10combined with the function of HarmOR10, we can determined the ligands of HarmOR10is benzaldehyde. We suspected benzaldehyde plays an important role to females in looking for host and spawning. But in electroantennography reaction, results showed that the response to benzaldehyde is males more sensitive than females. So we suspected benzaldehyde could have an impact on the feeding behavior of males.