Expression Characteristics Of The Circadian Clock Genes In BmN Cell Line

Circadian clock is a key regulation pathway in living organisms and it regulatsthe internal secretion, physiological metabolism and behavior from human tocyanobacteria, from organism to tissues and cultured cells. There are threecomponents in circadian clock: the input system, circadian oscillator and outputsystem. The cultured cells and tissues, although separated from the regulation of thecentral nervous system of the organism, also have the three components.The researchs about circadian clock in Bombyx mori almost focused on theindividual level during the past decades, and mainly explored the relationship betweendiapause and circadian clock. In this paper, the cultured silkworm cell line BmN wasused and the technological means such as qPCR, Western blotting,immunocytochemistry, RNAi were used to study the genes Cry1, Cry2, Per, Tim, Clkand Cyc in circadian clock regulatory network. Our research is almost about theexpression characteristics of the circadian genes after different light oscillation andtemperature oscillation treatment. The results are as follows:1. BmN cell include a endogenous circadian clock systemThe expressions of circadian clock genes Cry1, Cry2, Per, Tim, Clk and Cyc inBmN cell line can response to light and temperature stimuli, and can also response tothe light and temperature oscillation. CRY1, CRY2, PER and TIM proteins weredetected by polyclonal antibodies in BmN cell line, and the protein expression ofCRY1, CRY2, PER, TIM can also be influenced by light. These results implyed thatthe BmN cell line includes the endogenous circadian clock regulation system, andBmN cell line can be a model to study the circadian clock mechanism of the bombyxmori.2. Temperature has a compensation effect to circadian clock of the BmN cellline in the condition of constant lightThe expression levels of Cry1, Cry2, Per, Tim, Clk and Cyc genes of BmN cell line reduced significantly under the condition of24:0LD, and the expressionoscillation rhythm were also getting weaken or even disappeared. The expressionamount of Cry1, Cry2, Per, Tim, Clk and Cyc will get rose and the expression rhythmoscillations will also get restored when there’s a temperature oscillation added to theconstant light. The results showed that signal input pathways of the circadian clock inBmN cell line can response to the temperature oscillation, and the temperature has arole of compensation to circadian clock system of BmN cell line during the conditionof constant light.3. Cry1and Cry2genes have different response to light and temperature signalin BmN cell line.The gene expression of Cry1has a significant upregulation after the lightstimulation. Under different light rhythm, Cry1and Cry2are both very sensitive tothe change of light, the expression level of Cry1was significantly higher than Cry2(P<0.05); the moment of light-dark alternating, the expression of Cry1down-regulated,while the expression of Cry2did not change significantly. These results showed thatCry1can response to the light in the core oscillator regulated by light rhythm in BmNcell line and can be the signal receptor of the light. The gene expression of Cry2showed very significant change to temperature stimulation; while under thetemperature oscillation rhythm, oscillation rhythm of the expression of Cry1gotdisordered, while the expression of Cry2showed a timely response to temperaturechange and had a strong regularity of rhythm. We speculated that Cry2is the signalreceptor to rhythm regulation oscillator of temperature.4. The role of Per in the circadian clock loop of Bombyx mori may be greaterthan Cry1.Our laboratory analysed the structure of PER protein by bioinformatics means,found that PER and TIM protein of bombyx mori both has motif sites for proteinsignaling interaction, PER plays its role of information transfer in the form of proteinin the circadian clock loop. The earlier research of Cry1in our laboratory and theresults in this paper both shown that CRY1of Bombyx mori plays a important role asthe optical signal receptor at the individual level and the cellular level. This paperfurther interferenced anyone of Cry1and Per, the results found that the expression of the other gene will be significantly affected. when Cry1gene was interfered, only theexpression amount of Per and Cyc changed significantly; when Per gene wasinterfered, almost all genes been investigated undergone a significant change, theoscillation phase of gene expression was also changed, the oscillation rhythm becameweak. The results speculated that the Per gene plays a greater role than Cry1gene,there may be some genes in the BmN cell line to compensate the role of the Cry1gene, which shares the role of light signal receptor.