| Cotton bollworm (Helicoverpa armigera) is a dominant pest for agriculture in many countries. For insects, olfaction plays an essential role in processing chemical signals from the environment, leading to the detection of food, reproductive partners, oviposition sites, host, prey or predators. Odor detection is a complex procedure, in which many proteins are involved. Till now, some exciting progresses have been made in the olfaction recognition mechanism for insects, but the complete olfactory siganl transduction pathway is pooly understood. Elucidation of the olfaction signalling will provide the theoretical foundation for the development of the effective attractant and deterrent, and the fresh ideas and approaches for potent control of cotton bollworm, which can slower the revolution of its resistance to the Bt. cotton. In this paper, a Gq a subunit gene was cloned by RACE techniques and PCR. Temporal and spacial expression patterns of Gqa and SNMP in cotton bollworm were studied by semi-quantitive RT-PCR. Recombinant Gqa and SNMP were obtained by prokaryotic expression and purified by affinity chromatography. The interaction between the two cloned genes was determined by Yeast Two-Hybrid System and screening for related proteins to Gqo from H.armigera antennae-cDNA library were conducted with the same technique. The main results are as follows:(1) G protein a subunit was cloned for the first time from the antennae of Helicoverpa armigera by RT-PCR and (3'/5')-RACE techniques. It belonged to the Gaq family. The cDNA sequence was deposited in GenBank and the accession number is AY957405 and named as GqaHarm. The deduced amino acid sequence of GqaHarm shared high identity (>90%) with reported Gqa from other insects and invertebrates. The homology with Bombyx mori, its close sib population Mamestra brassicae, Drosophila melanogaster, Caenorhabditis elegans and Panulirus argus was up to 97.73%, 96.88%, 89.52%, 81.69%, 87.25% respectively.(2) Tissue-and stage-specific transcriptive levels were determined by semi-quantitative RT-PCR analysis and the results showed that Gqa was transcripted in multiple tissues and at different stages. Gqa was not only expressed in antenna, but also in the head without antenna, thorax, leg and wing of H. armigera. There was no obvious difference among the expression in the tissues. Moreover, it was also expressed in labial palpus, maxillary palpus and proboscis. Gqa was expressed in egg with the highest level, and in imago with the lowest level;and there was no significant diffenrence among the expression in different stages for the larva/and the pupae. The spacial and temporal expression pattern indicated that GqaHarm was a/ ubiquitous protein, which suggested that it might be involved in diverse signalingpathways.(3) Sensory neuron membrane protein (SNMP) of Helicoverpa armigera was reported by French Jacquin-Joly,E. and Francois,M.-C. in the year 2003 (GenBanK accession number: AF462067). A full-length cDNA encoding SNMP, was cloned by using the specific primers to the cDNA template from antennae of Helicoverpa armigera reared in our lab. The open reading-frame of SNMP was 1572bp, encoding 523 amino acid residues. There were two putative transmembrane domains, which is the typical characteristic of SNMPs. And it also shared high identity with sensory neuron membrane proteins from other insects. Semi-quantitative RT-PCR analysis showed that SNMP was not only expressed in antenna, but also in the head without antenna and leg of//, armigera. However, its expression in antenna was much higher than that in the head without antenna and leg. There was no obvious difference between the expression in the male and female antenna. Moreover, it was also expressed in labial palpus, maxillary palpus and proboscis which are the taste organs. SNMP was expressed in egg, the 10tb-day pupae and imago and expressed relatively low in egg. The spacial and temporal expression pattern indicated that SNMP may play roles in not only the odor detection but also the taste detection.(4) GqaHarm and SNMP genes were subcloned into prokaryotic expression vector pET-21b respectively. Bacteric expression vectors of target genes were successfully constructed and the target proteins were successfully expressed in E.coli through the inducement of IPTG. However , the expression product was insoluble inclusion bodies, which were solubilized and refolded . Then the recombinant proteins were purified by affinity chromatography. Pure Gqa protein was obtained , but SNMP was not purified successfully by the same purification method.(5) Interaction proteins were screened from the cDNA library of Helicoverpa.armigera antennae with cloned a subunit as bait protein by Yeast Two-Hybrid System. Forty-three positive clones were identified by SD/-Ade/-His/-Leu/-Trp/X-a-GaI and sequenced. Through GenBank blast, the cDNA inserts were highly similar to such mRNAs as Helicoverpa armigera pheromone binding protein (PBPHarm), Heliothis virescens pheromone binding protein-2 (PBPHvir2), Plutella xylostella glyceraldehyde-3-phosphate dehydrogenase, Spodoptera frugiperda ribosoma! protein S20, Helicoverpa armigera cytochrome oxidase subunit I gene, tRNA-Leu gene and cytochrome c oxidase subunit II gene,, Spodopterafrugiperda allatotropin neuropeptide precursor(AT2 gene), Bombyx^mori translationally controlled tumor protein (TCTP) and humoral lectin prepropeptfa/. Identification of these valuable clones provided substaintial foundation for furMef*. study of the function of G protein a subunit in signaling pathway of Helicoverpaarmigera.(5) The interaction between cloned Gq a subunit and SNMP was determined by Two-Hybrid System and no positive clones were obtained. One of the possible reasons is that proteins with weak affinities may escape detection or there is no interaction between the two proteins in Helicoverpa armigera. SNMP is presumed to interact with some a subunit of other G protein a subunit famlies, which needs the further studyIn this paper, G-protein a subunit was cloned for the first time from the antennae of Helicoverpa armigera. The accession number in GenBank is AY957405. The study on tissue- and temporal expression pattern of Gqa and SNMP indicated that G protein a subunit was a ubiquitous protein in Helicoverpa armigera and might be involved in diverse signaling pathways. The temporal and spacial expression pattern of SNMP suggested that SNMP was not antennae-specific, which was involved in both olfactory detection an taste function. These results of two-hybrid screening indicated that G protein a subunit could directly interact with pheromone binding protein, which had some roles in odor signal transduction. G protein a subunit could not interact with SNMP in the two hybrid system.There is no internal and oversea publication concerned with above studies on G proteins in Helicoverpa armigera. These achievements provide substaintial foundation for further research of G proteins and G-protein signaling pathways in cotton bollworm. It also has important theoretic and practical significance on going deep into interaction between insect and host plant, pest-resistant mechanism of plant and exploring new control method.
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