Analyzing The Promoters Of CYP9A19and CYP9A22Genes In The Silkworm, Bombyx Mori

Cytochrome P450is an important metabolic enzyme which has been found in all livingorganisms, including plant, animal, fungus and bacterium. They have important roles inthe detoxification of xenobiotics, such as pesticides, plant allelochemical andanthropogenic pollutants. The silkworm Bombyx mori is not only a model organism forlepidopteran insects, but also economically important in silk industry. And they are thematerials of bioreactor and new insect industry. To further investigate the inducedexpression and promoter activity of CYP9A19and CYP9A22in silkworm, the presentstudy were addressed. The main results were shown as follows:We measured the transcription levels of CYP9A19and CYP9A22genes by dualspike-in qPCR in each tissues of Bombyx mori with no induction or NaF induction. Theresults show that, CYP9A19gene had high expression levels in the midgut, silk gland, fatbody and malpighian tubule. CYP9A22gene had expression levels in the midgut, silk glandand malpighian tubule, but no expression in fat body. The transcription profiles after NaFinduction were tissue-specific. The induction in the midgut and fat body is called the positiveinduction which is up-regulated then recovered, and that in the malpighian tubules is calledthe negative induction which is down-regulated then recovered. The results showed thatCYP9A19and CYP9A22genes have closely relationships with metabolism of NaF insilkworm.At the cellular level, the RNAi experiments were conducted. Bm-CYP9A19-663andBm-CYP9A22-1384highly reduced the expression levels of CYP9A19and CYP9A22to49.59%and48.00%of its normal levels respectively. CYP9A19expression significantlydecreased after CYP9A22expression was inhibited, while CYP9A22expression wasunchanged when CYP9A19expression was inhibited. This demonstrated a compensationfrom CYP9A19after loss of CYP9A22.The overexpression of CYP9A19and CYP9A22genes have a relationship with thedetoxification of xenobiotics. In order to study the mechanisms underlying regulation ofCYP9A19and CYP9A22, functional analysis of the promoters of CYP9A19and CYP9A22 from Bombyx mori was performed in this study. Luciferase reporter plasmids containingserially truncated promoter fragments of the two genes were transiently transfected intoBmN cells with internal control vector pRL-TK. The promoter activity was measured withdual-luciferase reporter assay system. Functional analysis showed that the regions-1496to-1102for CYP9A19, and-1630to-1210for CYP9A22were essential for basaltranscriptional activity. After BmN cells induced by0.1×10^-2,0.1×10^-3,0.1×10-4mol/LNaF,0.1×10^-3mol/L NaF induction significantly increased the activity of the two CYP9Apromoters. The overexpression of CYP9A19and CYP9A22genes after NaF inductionmight have a relationship with the upregulation of the promoter activity. This study wouldprovide an important foundation for investigating transcriptional regulation mechanisms ofCYP9A19and CYP9A22in Bombyx mori. However, the precisely funcion and effectmechanisms needs further investigaiton and study.Dual luciferase reporter assay system is widely used in promoter analysis. We haveconducted an in-depth analysis of dual-luciferase reporter assay on vector sizes andconstruct protocols. The results showed that the larger the vector, the lower thetransfection efficiency. We obtained a standard curve and proposed a normalizationmethod that minimize transfection efficiency errors. On the other hand, we studied thepromoter activities of CYP9A19and CYP9A22gene by two protocols. Protocol1insertedthe promoter into the MCS region according to the manufacturer’s instructions. Protocol2inserted the promoter directly next to the translation start of the luc+gene. Theexpression profiles from the two protocols were in substantial agreement althoughexpression using protocol1was nearly twice as high as protocol2. In other words,protocol1was a better reflection of the expression profiles, although protocol2was abetter reflection of the true value of the promoter activity. This study on these two issueswould provide an important foundation for ease and accuracy of future experiments.