Study On Regulation Of Detoxification Gene Expression In Silkworm, Bombyx Mori

In nature, in order to meet its own growth, development and survival needs,organisms make material exchanges with the external environment all the time. Some ofthese substances, in order to meet the organism nutrition requirement, is beneficial to theorganism, and some is harmful to them. In order to survive, the organism must have somephysiological, biochemical, and even behavioral adaptability. Catalyzing and decomposingmetabolites or heterologous material through the body’s detoxification enzyme systemsplay a major and common adaptation mechanism. Insect detoxification enzymes are a classof heterogeneous enzymes which can metabolize endogenous or exogenous substrates,these detoxification enzyme mainly include: cytochrome P450, glutathione-S-transferaseand carboxylesterase.In order to obtain a stable reference gene, standardization of reference gene by usingthe geNorm and NormFinder Software analysis is the key step in the process of relativequantification. However, the error caused by the relative quantification have not beeneliminated even choose a relatively stable reference gene. By adding an external referencegene into the sample, the target gene expression is calibrated and tracked, more accuratecorrection results are obtained by using Dual spike-in qPCR. However, according to theprinciple of the method, the workload is increased, and to some extent the experimentalcost and time is increased too. To solve this problem, we envision a new experimentalstrategy that we can quantify a standardized reference gene firstly by using Dual spike-inqPCR, and then use this gene to correct the target gene expression. The result showed thatthis strategy not only ensured the accurate results, but also reduced the experimental costand time. So, it promoted the further development and innovation of quantitative PCR tosome extent.In order to understand the distribution of main detoxification genes in silkworm andthe impact of exogenous material on the expression of detoxification genes, thetranscription levels of detoxification genes in normal silkworm and silkworm fed by ecdysone and rutin were measured by using Dual spike-in PCR. The results show that lessgenes are involved in ecdysone metabolism in the midgut, and BmGST genes are maininvolved in rutin metabolism. The main genes involved in ecdysone metabolism areCYP9a20, BmGSTo1, BmGSTz2in fat body; more genes are involved in rutin metabolismsuch as BmGST genes. The main genes involved in ecdysone me tabolism are CYP6ab4,BmGSTs2, BmCarE-15in malpighian tubules, and BmGSTo1is mainly involved in rutinmetabolism.To further understand the physiological functions of the detoxification genes, wedesigned three different siRNA for each gene in the present study, and the siRNAfragments with the most significant effect are filted. The results show that theCYP6ab4-928，BmGSTd1-405，BmCarE-10-519are most effective, the transcription levelof each gene decreased to26.5%,39.6%and17.9%respectively, so they can be used forfurther in vivo RNAi experiments.In order to understand the impact of RNAi on silkworm gene expression in vivo, theeffective siRNA were injected into fifth instar silkworm larvae, and then we measured andanalyzed the changes of transcription levels of detoxification genes in mid gut, fat bodyand malpighian tubule by using real-time PCR. Experimental results show that theexpression of three measured genes can successfully be suppressed, and the extents are notsame. The effect at48h after injection is more obvious than24h. We surveyed thecumulative mortality rate of silkworms after feeding phoxim, and the results show that themortality was significantly increased to88%,84%and89%, respectively at48h afterinjection.For the study on the mechanism of expression and regulation of main detoxificationgenes, we analyzed the function of promoter region of CYP6ab4, BmGSTd1andBmCarE-10genes, and constructed recombinant plasmids containing the luciferasereporter gene and promoter. The internal control vector pRL-TK was co-transfected intoBmN cell with recombinant plasmids, promoter activity was detected by using dualluciferase reporter gene assay system. The results show that the promoter activity ofCYP6ab4and BmGSTd1genes were raised by ecdysone, and rutin can raise the promoteractivity of BmGSTd1and BmCarE-10genes. The promoter activity area of CYP6ab4,BmGSTd1and BmCarE-10gene exists in the respective promoter5’ unilateral-827~-722bp,-1385~-1299bp and-705~-625bp of various regions respectively, and the presence of the BR-C Z, Kr and Sn may play an important role in the process of gene expressionregulation.We use rAcMNPV as a carrier of the gene-mediated fluorescence quantitativeexpression vector system to analyze the functions of the detoxification gene promoterregion. Constract a dual fluorescent plasmid of FHNLuc-A3RL, in which Fluc gene isdriven by detoxification gene promoter, and Rluc is driven by A3promoter. We preparedrecombinant baculovious through the Bac-to-Bac system, and injected them into fifth instarsilkworm, and analysis the luciferase activity. The results show that the of activities ofthree gene promoters in malpighian tubule and fat body are higher than in other tissues,and the promoter activity area is consistent with the measured results in BmN cells.This study analyzed the whole basic transcription situation of detoxification genes insilkworm by using real-time PCR, and analyzed the promoter sequence of three importantdetoxification genes by using dual luciferase reporter gene assay system. Importantregulatory regions and associated protein factors were obtained. This study providedimportant theoretical basis for the study of the regulation of gene expression in silkwormdetoxification enzymes.