RNA interference to investigate the function of target of rapamycin (TOR) gene in Bemisia tabaci (Gennadius)
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Date
2020
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Punjab Agricultural University, Ludhiana
Abstract
The target of rapamycin gene was amplified in ten overlapping fragments from whitefly. The full length of TOR gene resulted in 7311 bp nucleotides and 2437 amino acids. TOR gene showed 98.66 per cent homology with predicted sequence of Bemisia tabaci serine/threonine-protein kinase mTOR. Ninety-five nucleotides were found to be different from the predicted sequence of TOR gene in B. tabaci, but only two amino acid differed. Amino acid at position 1448 i.e. asparagine replaced threonine and at position 1768, arginine replaced tryptophan in sequenced TOR gene as compared to predicted TOR amino acid. Phylogenetic analysis of amino acid sequence revealed that TOR gene of B. tabaci, Acrythosiphum pisum and Nilaparvata lugens were more closely related to each other and grouped in one cluster. dsRNA corresponding to TOR (@1.0, 0.5 and 0.1 μg/μl) and gfp (@1.0, 0.5 and 0.1 μg/μl) gene were synthesized and fed to the whitefly adults to record the adverse effect on biology of whitefly. Continuous feeding of dsRNAs to whitefly adults showed that maximum mortality (47.75 %) occurred after 48 hours of feeding, when whiteflies were fed with dstor @ 0.1 μg/μl, which was statistically at par with all the treatments of dstor and significantly higher than dsgfp treatments and control. The bioassay after feeding of whiteflies for 48 hours on artificial diet was conducted three times in June-July, July-August and August-September, 2019. The adult mortality after 48 hours of feeding was significantly higher in dstor fed whiteflies than control and dsgfp treatments. The maximum mortality (46.66, 41.66 and 26.25 %) were recorded in dstor @ 1.0, 0.5 and 2.0 μg/μl in all the experiments, respectively and the minimum mortality (16.66, 10.00 and 7.5%) was recorded in dsgfp @1.0 μg/μl, 1.0 μg/μl and control, respectively. Significant effect of silencing of TOR gene was recorded on fecundity of B. tabaci fed on dstor in two experiments, which was statistically lower than dsgfp and control. Minimum egg laying (41.25 and 30.33 per female) were recorded in dstor @ 1.0 μg/μl, however maximum egg laying were 70.00 and 64.77 per female in dsgfp @ 0.1 μg/μl in both the experiments. Nymphal mortality increased after silencing TOR. It was significantly higher when whiteflies were fed with dstor @ 2.0, 1.0, 0.5 and 0.1 μg/μl as compared to all the concentrations of dsgfp and control in all the three experiments. The major significant difference with respect to nymphal mortality was observed only during first instar, however nymphal mortality in second and third instars were statistically non-significant. No significant effect of silencing of TOR was observed in case of nyphal duration, pupal mortality, pupal duration and adult emergence. Upon feeding of dstor to adult whiteflies for 48hours, RNAi experiment showed that 55.34, 57.21 and 63.36 per cent expression of TOR gene in dstor @ 1.0, 0.5, 0.1 ug/μl, respectively as compared to dsgfp @ 1.0 ug/μl (100 %). After 96 hours, feeding mRNA level of TOR gene was 13.77, 16.52, 24.07 and 28.75 per cent, when whitefly adults were fed at dstor @2.0, 1.0, 0.5 and 0.1 ug/μl as compared with dsgfp @ 1.0 ug/μl (100 %). These qPCR experiments confirm the partial silencing of TOR gene in whiteflies when fed with dstor.
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