Function Analysis Of InaC Gene In Light Responses Of Bactrocera Dorsalis

The oriental fruit fly, Bactrocera dorsalis(Hendel), creats huge economic damage to fruit and vegetable production, because of the wide host plant range, liking lay eggs in the fruit and sneaking into the internal by larvae. Colored papers’ traps and light trapping are safe and harmless control methods to environment and easy to operate, based on the study of visual behavior of oriental fruit fly. The protein kinase C(PKC) is expressed exclusively in the fruit fly eyes, it involves in many processes include signal transduction and light adaptation, and it activates the rhodopsin into metarhodopsin. However, it’s not been reported whether eye-PKC influence the biological behavior of light response in oriental fruit fly.In this paper, we chose the ina C, the gene encoding eye-PKC protein, as the object of the study. RNAi technology and real-time quantitative PCR and other molecular biology techniques were employed to study the gene expression profiles in different developmental stages and different tissues.We studied the role of ina C in light responses by observing the phototaxis and mating behavior of Bactrocera dorsalis. The result has not only laid a theoretical foundation, but also provides a reference for the prevention of Bactrocera dorsalis. 1. Cloning of Bactrocera dorsalis ina C gene and Analysis of gene expressionpatterns.We cloned the full sequence c DNA of ina C gene from B.dorsalis. The ORF nucleotide sequence contains 2052 bp, encoding a 683 amino acids. The structure domain prediction of gene by SMART showed that eye-PKC in B.dorsalis, was similar to INAC encoded by ina C in Drosophila, containing two PKC conserved domains conserved domain C1, one PKC conserved domains conserved domain C2, a S_TKc domain and a S_TK_X domain. The results of the homology comparison showed the ina C amino acid sequence was very conservative between affinis insects, being 97% compared with Bactrocera cucurbitae and Ceratitis capitata, and 84%, 83%, 82% compared with Drosophila melanogaster, Musca domestica and Calliphora vicina respectively. Phylogenetic tree was made with Bactrocera dorsalis ina C gene and other similar eye-PKC amino acids, the result was consistent with the homology alignment. From the near to the distant of evolution were B.cucurbitae, C.capitata, D.melanogaster, M.domestica and C.vicina.We detected the B.dorsalis ina C gene expression profiles in different developmental stages and different tissues by q RT-PCR technology. ina C is hardly expressed in eggs and larvals, in pupae it expressed at a low level, ina C had a high expression level in adults, and the expression of mature adult was nearly 1.75 times compared with adults at the beginning of emergence. In different tissues, ina C gene is highly expressed in head, and almost not expressed in other tissues. 2. Function of ina C in phototactic behaviorE.coli stain HT115 was engineered to expressed ina C double-strand RNA, then we silenced gene by feeding bacteria expressing ds RNA in B.dorsalis. The result of q RT-PCR of ina C expressed in head showed ds RNA could cause a systemic effects of interference in B.dorsalis. Compared with the control group, the gene expression of ina C ds RNA treatment group declined by 54%.To test the differential phototaxis between two different wavelength light, we used a Y-maze apparatus. In a “violet vs other color(red, orange, green, blue)” or “yellow vs other color(red, orange, green, blue)”, flies preferred violet and yellow, but there’s no differeces between violet and yellow in “violet vs yellow”. After knocking down ina C, the number of flies on the side of the violet showed a decrease in the “violet vs red or orange or green” choice paradigmis(the choosing rates reduced to 55%, 54.43%, 59.74% from 75%, 80.77%, 81.16%). the number of flies on the side of the yellow also showed a decrease in the “yellow vs red or orange or blue”(the choosing rates reduced to 59.42%, 51.35 %, 47.14% from 75.32%, 71.05%, 71.25%). The control showed no differential attractiveness between yellow and purple in “violet vs yellow”(the attraction rate of yellow and violet were 48.75% and 51.25%), but in the ina C treatment group, these flies showed a preference for yellow(80.88%) over violet. The results suggest that the preference for violet or yellow was smaller after interfering inaC gene, by measuring differential phototaxis between spectrally different lights, and the effect was more remarkable on the violet. 3. Function of ina C in mating behaviorIn this study, we found mating is nearly absent in the dark, and the rate of mating success was low(45%) under white light within 1 hour, in color light the mating rate were higher, all of them reached 80%. The courtship time were longer in yellow or green than in red, orange, blue or violet. The rates of mating success were about 80% in red, orange, blue or violet, but in yellow or green it just about 50%. After interfering ina C gene, the rates showed a significant decreased in green or violet condition compared with control(green, the rate of egfp ds RNA group was 95%, the rate of ina C ds RNA group was 50%, χ2=10.16, df=1, p<0.01; purple, the rate of egfp ds RNA group was 100%, the rate of ina C ds RNA group was 60%, χ2=10.00, df=1, p<0.01), there were no significant differences in the other colors conditions. The courtship time extended under orange, yellow, green, purple light conditions after decreasing ina C expression. The number of mating successfully became smaller within 30 minutes(compared with control, the significance were orange: χ2=5.01;7 yellow: χ2=6.47, p<0.05;7 green: χ2=8.29, p<0.01;7 purple: χ2=7.03, p<0.01).