| Cytochrome P450 is an important metabolic enzymes, which is involved in the synthesis and decomposition of exogenous and endogenous compounds. Cytochrome P450 has a variety of functions in the insect's life, such as participation in the metabolism of pesticides and plant secondary, and the synthesis of ecdysone, juvenile hormone, sex pheromones. At the same time, cytochrome P450 has a closely relationship with the growth, development and defense of insect. We studied the functions and regulation mechanism of the CYP 3 and CYP 4 family genes in silkworm in order to research the relationship cytochrome P450 and metabolism of substances in vivo. The results are listed as follows:We measured the transcription level of CYP3 family by dual spike-in qPCR in midgut and fat body after induced Bombyx mori with ecdysone(2×10-3μg/μL). The results show that, the transcription level of CYP302, CYP306 and CYP339 were increased respectively by 191.4 times, 7.4 times and 421 times in fat body, the level of the remaining gene transcription induced by the ecdysone did not change significantly or undetectable the transcriptional activity. That CYP339 gene may be involved in the metabolism of silkworm ecdysone. This work layed a theroy foundation for further study the relationship between P450 genes and the metabolism of endogenous substances .We chosed two genes (CYP306, CYP339) and studied the mechanisms underlying regulation of these genes which related to the metabolism of ecdysone. In order to study the mechanisms underlying regulation of CYP306, CYP339, functional analysis of the promoter of CYP306, CYP339, from Bombyx mori was performed in this study. Luciferase reporter plasmids containing serially truncated promoter fragments (2000bp, 1600bp, 1200bp, 800bp, 400bp) of the two genes were transiently transfected into BmN cells with internal control vector pRL-TK under the mediation of liposome. After in vitro induced by ecdysone, the promoter activity was measured with dual luciferase reporter assay system. Functional analysis showed that all the deleted fragments of the two genes had promoter activity expect for pGL3-306-3, pGL3-306-4, pGL3-306-5, and a fragment of 1600 bp exhibited the highest level of promoter activity. Transcription enhancer elements may exist in the region between 1200bp lenth 1600bp lenth of regulation area, and transcription repressor elements may situate in the upstream of the two regions. Ten deletion fragments of the two genes induced changes in activity mixed, after BmN cells induced by 2×10-2, 2×10-3, 2×10-4μg/μL solution of ecdysone. 2×10-3μg/μL induce the most significant increase and the maximum was up to 10 folds than that of pGL3-Basic. In summary, overexpression of CYP306 and CYP339 genes might have a relationship with the upregulation of the promoter activity. This study would provide an important foundation for investigating transcriptional regulation mechanisms of CYP306 and CYP339 overexpression in Bombyx mori. However, the precisely funcion and effect mechanisms needs further investigaiton and study.The present study was undertaken to clarify the change of induction of CYP4M5 and CYP4M9 expression level by rutin. In this study, we used dual-spike-in qPCR to examine expression profiles of the CYP4M5 and CYP4M9 genes in the larval midgut and fatbody after exposure to rutin. In organization-course study, rutin at middle concentration (5×10-2 ng/μL) caused significant upregulation of CYP4M5 and CYP4M9 genes at early time point (2 h) in fatbody, higher concentration (5×10-1 ng/μL) did secondly, while lower concentration (5×10-3 ng/μL) caused little change. In the midgut of silkworm, rutin in all concentrations didn't affect the expression of the two genes. These findings showed that induction of CYP4M5 and CYP4M9 expression level by rutin are concentration depended and tissue-specific. Collectively, experimental results showed that P450 4 family genes have closely relationships with metabolism of rutin in silkworm. |
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