Gene Cloning And Expression Analysis Of Odorant Receptor And Arginine Kinase Gene From Helicoverpa Assulta (Guenée)

Abstract:Insect olfactory system is a highly specific and extremely sensitive chemical detector,and can identify the specific odor molecules from the environment, by which insects can function as feeding. calling behavior, selection of the host and oviposition site, avoiding of predators. The odor molecule can be detected by binding with its receptor. Therefore studies on odorant receptors play a great significant role in elucidating the molecular mechanism of the smell identification. Besides, energy metabolism is a foundation for insect odorant perception, among which arginine kinase plays an important role in insect metamorphosis process. The research on both (odorant receptors and argentines kinase) will provide foundation to understand molecular mechanism of odorant receptor recognition ordor in insects. The main contents are as follows: Molecular cloning and tissue-specific expression of odorant receptors from the Helicoverpa assulta (Guenée), Cloning and mRNA expression analysis of arginine kinase gene from the Helicoverpa assulta (Guenée). The main results are as following:(1)A cDNA sequence encoding an odorant receptors protein from antenna of Helicoverpa assulta was obtained by reverse transcription polymerase chain reaction ( RT-PCR). Sequences analysis results showed HassOR18 open reading frame (ORF) was 1197 bp in size, encoding 399 amino acid residues with the predicted 46.6 kD molecular weight (MW). The predicted protein obtained, containing seven putative transmembrane domains, shares more 80% amino acid identity with other Lepidoptera moth homologs. This sequence, named as HassOR18, has been deposited in the GenBank database with accession number HM750915.(2)The tissular expression of HassOR18 was analyzed by using real-time PCR. The results show that HassOR18 transcript was detected in the antennae of both male and female adults and female Proboscises. Moreover, the HassOR18 transcript was expressed richly in male antennae, compared with the female antennaes and female Proboscises.(3)Helicoverpa assulta arginine kinase cDNA was isolated by using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA 3'ends (RACE). Nucleotide sequence analysis revealed that the cDNA fragment contains a 1068 bp coding region and 238 bp 3'untranslated region (UTR). Sequencing analysis further showed that predicted amino acid sequence contains three conserved domains: CPTNLGT which is Catalytic activity, Asp(61) and Arg(192). Amino acid residues 61 and 192 play the key role in regulating the synergism of substrate binding inoyster arginine kinase.The predicted protein obtained shares more 70% amino acid identity identity with other known arginine kinase. This sequence has been deposited in the GenBank database with accession number HQ336337 and named as HassAK.(4) The temporal and tissular expression of HassAK transcript was studied by using Quantitative PCR.The results showed that HassAK reach expression peak in pre-pupa and 4th-instar larvae, consistent with the titer changes of 20E, molting hormone in development stages. Real-time PCR analysis revealed that HassAK could be detected in head, midgut, fat body, epidermal tissue and abdominal leg of larva. Among them, HassAK was richest in abdominal leg and midgut.(5) The effects of high and low temperatures on HassAK expression were analyzed in fat body of 5th-instar larvae by using Quantitative PCR.The result revealed that high and low temperatures can be up-regulated the expression of HassAK, which indicated that HassAK gene maybe take part in insect resistance to adverse external environmental.