| Bombyx mori is one of model organisms in Lepidoptera, rutin is one of plant secondary metabolites, using detoxification enzymes to etabolize it is an important way for insects adapt to plant secondary metabolites. This research take the Bombyx mori as the object of study, through the current real-time quantitative PCR study of silkworm commonly used endogenous reference gene analysis, and compare with the dual-spike-in qPCR method. Using this new method, has conducted the induction of expression of glutathione-S-transferase genes in Bombyx mori by rutin. The main results are as followed.1. Comparing the both method of normalization in real-time quantitative PCRIn this study, we detected transcription levels of BmGSTd1 in midgut and fat body from fifth instar Bombyx mori larvae treated with of rutin (5×10-2 ng/μL). In order to select suitable reference gene for the induction experiments in B.mori, the transcription levels of BmGSTd1 in different tissues of B.mori treated with rutin were detected, with two housekeeping genes (Actin3 and GAPDH) and joining an exogenous gene (IFP2) for reference to normalize the data. Then, the transcription levels of two housekeeping genes in midgut and fat body of fifth instar B. mori larvae and their responses to rutin were investigated. The results showed that both transcription levels in different organizations are not constant, and change a lot after induction. The results showed that expressions of housekeeping genes were not constant after induction, while exogenous gene, rise superior to experimental conditions, can guarantee the accuracy of the results. In B. mori, joining exogenous genes to normalize the qPCR data is the suitable method in induction experiments to test the expression of genes. 2. Induction of expression of glutathione-S-transferase genes in Bombyx mori by different concentrations of rutinWe detected the transcription levels of omega-class GSTs genes in midgut and fat body of the fifth instar larvae and those treated with different concentrations of rutin using dual-spike-in qPCR. The results show that the rutin (5×10-2 ng/μL) can induce a significant change of the transcription levels of GSTs genes. In the midgut, compared with the control, the transcription levels of BmGSTo1, BmGSTo2 and BmGSTo3 increased by 7 141,1 391 and 3 398 times in midgut at 24 h after rutin induction; while in fat body, the transcription levels of BmGSTo1 was the highest and arriving the peak value after being induced 2 hours. Compared with the treatment with 5×10-2 ng/μL rutin, 5×10-1 ng/μL rutin could induce a little change of the transcription levels of GSTs genes in fatbody and the time when the expression peak occurred was not the same. However, 5×10-3 ng/μL rutin could not induce the change of the transcription level of the GSTs genes.3. Induction of expression glutathione-S-transferase in Bombyx mori by rutinWe detected the transcription levels of GSTs genes in different tissues of the 5th instar larvae and those treated with rutin (5×10-2 ng/μL) using dual-spike-in qPCR. The results showed in the midgut, BmGSTs1, BmGSTz2, BmGSTe1, BmGSTe2 and BmGSTe6 had higher transcription levels and reached the maximum at 24 h after induction; in fat body, all of genes except BmGSTe2 and BmGSTe5 had higher transcription levels and reached the peak at 2 h and 4 h after induction; while in malpighian tubules, only can detect low-level transcription of BmGSTs1, the transcription level of other GSTs genes did not change significantly or was undetectable in. The results suggest that the GSTs genes play an important role in metabolism of rutin, will provide reference for research of the plant secondary metabolites influence on the sensitivity of silkworm resistance to pesticide. |
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