Identification And Cloning Of Antennal Olfactory Related Genes Of Anoplophora Glabripennis (Coleoptera: Cerambycidae)

The Asian long-horned beetle, Anoplophora glabripennis (Coleoptera: Cerambycidae) is one of the most important insect pests and the great losses have been caused by it on the greening and forest construction project in China. Olfaction plays a critical role for the insect detecting odors, seeking host and mating. Based on the study of antennal sensory organs, it’s possible and necessary to study the molecular mechanism of olfactory recognition in order to understand how insect can distinguish different chemicals. Moreover, elucidating the molecular mechanism of insect olfaction can offer a theoretical foundation in developing safe, effective attractant or deterrent to control insect pests. Meanwhile, insects as ideal model organisms, study on their olfactory can help us to understand the envolution of human olfaction.In this study, I used differential display reverse transcription PCR (DDRT-PCR), annealing control primer (ACP) technology and rapid amplification of cDNA ends (RACE) method to clone the olfactory related genes in the antennae of A. glabripennis and analyzed their possible structures. The results are as follows:1, High quality RNA was isolated from antennae and deantennated body of A. glabripennis by TRIzol method, then purified and mRNA was isolated from them. Samily with drosophila, there were much more 18S rRNA than 28S rRNA in the total RNA. It might be that the 28S rRNA was cut into two fragments which had almost the same molecular weight with the 18S rRNA.2, To study the antennae differentially expressed genes, a mRNA differential display PCR system which was suitable to study the A. glabripennis was established. Antennae differentially expressed cDNA fragments were generated by RT-PCR and separated on 6% denatured polyacrylamide gel electrophoresis and subjected to reverse Northern blot hybridization reducing false positive fragments, then cloned differential cDNA fragments into pMD20-T vector and sequenced. The cDNA fragments’ sequences were compared with data in GeneBank using basic local alignment search tool (blast). 28 differentially expressed cDNA fragments were obtained and 14 of them were highly homologous with known functional gene products, 4 fragments were hypothetical proteins and the others had no information found in Genebank.3, In order to identify special expressed genes in antennae of A. glabripennis, a novel annealing control primer(ACP) system was adapted to identify differentially expressed genes. By using 120 pairs of ACP primers, 87 differentially expressed cDNA fragments were obtained and 28 fragments were highly homologous with known functional gene products. In this 87 cDNA fragments, 9 of them were also found by mRNA differential display PCR system.4, Two putative odorant binding proteins (OBPs) and one putative chemosensory protein (CSPs) were identified and cloned. The putative OBPs and CSP were identified by sequence alignment. Gene-specific primers for RACE were designed according to the cDNA fragments’sequences and used in polymerase chain reaction in order to obtain full-length sequences. The proteins Agla-OBP1, Agla-OBP2 and Agla-CSP1 encode 134, 128 and 123 amino acid deduced sequences, respectively. All of them have signal peptide in their N-terminal. Database searches suggest that the Agla-OBP1 and Agla-OBP2 are homologs of OBPs from other insects and Agla-CSP1 shares a high level of identity with previously described CSPs.