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目的本研究试图证明,特异性腺苷酸环化酶激活剂forskolin(FSK)可以通过调控FMR1启动子区内的MSE/CRE位点而再激活封闭的FMR1基因。方法拟以FMR1启动子MSE/CRE位点为研究对象,在不改变MSE的整体结构功能的情况下,运用分子生物学方法诱导CRE位点突变,达到其灭活CRE功能目的,从而建立正常MSE以及两种CRE突变启动子M1、M2双荧光素酶报告基因系统,比较CRE在正常或灭活情况下,FSK药物对FMR1诱导效果的影响。结果成功构建FMR1启动子区MSE/CRE位点双荧光素酶报告基因系统,结果显示当CRE位点正常时,FSK对活性低下的FMR1启动子有显著地激活作用,而当CRE位点突变之后,FSK则无此作用,由此证实CRE位点是FSK重启FMR1基因的关键位点。结论提示腺苷酸环化酶激活剂FSK可能主要是通过FMR1启动子区内的MSE/CRE重叠位点,经c AMP通路实现了对FMR1基因的调控。
This study was designed to demonstrate that forskolin (FSK), a specific adenylate cyclase activator, can reactivated the closed FMR1 gene by regulating the MSE / CRE locus in the FMR1 promoter region. METHODS: The site of MSE / CRE of promoter of FMR1 gene was studied. Without changing the overall structural function of MSE, molecular mutagenesis of CRE site was induced by molecular biology to achieve its purpose of inactivating CRE function, thereby establishing a normal MSE As well as two CRE mutant promoters M1 and M2 dual luciferase reporter gene systems were used to compare the effect of FSK on the induction of FMR1 under normal or inactivated conditions. Results The dual luciferase reporter system of MSE / CRE locus in FMR1 promoter region was successfully constructed. The results showed that when CRE site was normal, FSK had a significant activation on the less active FMR1 promoter. When CRE site was mutated , FSK does not have this effect, thus confirming that the CRE locus is the key point for FSK to restart the FMR1 gene. The results suggest that the adenylate cyclase activator FSK may be mainly through the FMR1 promoter region MSE / CRE overlapping sites, through the cAMP pathway to achieve the FMR1 gene regulation.