Effect Of Temperature To The Expression Of Circadian Clock Genes In BmN Cell Based On Signal Pathway Of Light Zeitgeber

Biological rhythm exists widely in the biosphere, from prokaryotic unicellular tohigher plants and animals, including human life activities being under the control ofcircadian clock. The circadian clock system consists of input channel of zeitgeber signal,core oscillator and output channel of zeitgeber signal. Although cells and tissues are outof the regulation of organism central nervous, their internal peripheral circadian clockalso have endogenous rhythms.Many physical activitives and behaviors of insect, such as hatching, feathering,feeding, metabolism, mating, moulting, even the immune and learning memory showsome biological rhythm. Light, as the most important zeitgeber of insects, is deeplystudied in Drosophila melanogaster and other model insect on the aspect of the inputpathway and the feedback loop, but the action mechanism of temperature zeitgeber hasnot been elucidated. The important reason is lack of effective biological model. On thebasis of light zeitgeber signal loop, this study uses the technology of qRT-PCR, Westernblotting and immunofluorescence to study the expression features of rhythm oscillationof circadian clock gene Cry1, Cry2, Per, Tim and the corresponding protein in silkwormBmN cell under different temperature cycle and high temperature stimulation treatment.The results are as follows:1. Subcellular localization of silkworm circadian clock proteinOn the condition of26℃,12h L:12h D, the expression of circadian clock proteinCRY1, CRY2, PER and TIM could response to light stimulation, and had a rhythmoscillation for light. The results showed that under the circumstances of12h L:12h D,CRY1did not appear in the nucleus, only existing in the cytoplasm; CRY2stablyexisted in the cytoplasm, and got into the nucleus by a little amount; PER existed both in cytoplasm and nuclei; TIM got into the nucleus when exposed to light and returnedfrom nucleus to cytoplasm in darkness. We speculated that as optical signal receptor,CRY1passed the stimulation to CRY2, PER and TIM. Then, these three might combineto play a role in transcriptional suppression in the nucleus and TIM might function as anegative feedback signal in the cytoplasm.2. The influence of37℃instantaneous high temperature stimulation on thecircadian clock gene expression in BmN cellsIn order to verify whether the circadian gene response to temperature signal, weinvestigated the negative feedback signal pathway of light zeitgeber core circadianclock gene expression of Cry1, Cry2, Per and Tim in48h after the instantaneous hightemperature stimulation, with the method of30min’s37℃instantaneous hightemperature stimulation. It turned out that the stimulation of37℃instantaneous hightemperature could induce the expression rhythm oscillation of circadian clock genes,characterized by the increase of expression quantity and enhancement of volatility. Theexpression amplitude of Cry1, Cry2and Per were significantly changed, while Tim hadno significant change. Cry2was characterized by phase advance and a longer period ofoscillation while Cry1’s became shorter. We speculated that Cry1and Cry2might be themajor genes which responded to temperature zeitgeber. The effect of Per might berelatively weak, and Tim had no direct relations to the signal response of temperaturezeitgeber.3. The expression characteristics of circadian clock genes and proteins inBmN cell under the circumstance of temperature rhythmShu MeiYing’s research (2013) has shown that the expression of circadian clockgene Cry1, Cry2, Per and Tim have under LL,2℃oscillation temperature rhythm (12h26℃:12h28℃)?. This investigation, with the method of Western blotting, furtherfound that at the protein level, only content of Cry1had rhythm oscillation while Cry2,Per and Tim had no consistent rhythm oscillations with gene expression. We speculatedthat2℃temperature difference was not enough to cause the response of protein to thetemperature signal in silkworm BmN cells.Under LL,5℃oscillation temperature rhythm (12h20℃:12h25℃or12h25℃:12h30℃), the investigation further showed that Cry1and Cry2were sensitive tothe temperature response. While under DD, as the same as oscillation temperature rhythm of LL, it showed that Cry1and Per were the major response role. We speculatedthat the existence of light might affect the function of temperature. On that basis, wechose DD,5℃oscillation temperature rhythm (12h20℃:12h30℃) to evaluate theresponse of gene and protein to temperature oscillation. The outcome demonstrated thesynchronization of rhythm oscillation between circadian clock gene and protein andCry1(CRY1), Per (PER) were sensitive to temperature change, Cry2(CRY2)responding weak while Tim (TIM) showing no change.According to above results2and3, the conclusions can be drew that Cry1and Perwere sensitive to stable temperature rhythm, while Cry2might be more sensitive to hightemperature for a short period of time. We speculated that Cry1was the most activemember of the temperature zeitgeber signal pathway in BmN cell.