【作者】 杨洋；

【导师】 黄瑾； 曾妍；

【作者基本信息】 石河子大学 ， 生物化学与分子生物学， 2017， 硕士

【摘要】 目的:本次实验研究主要探讨去泛素化酶OTUD5对PIF1蛋白的调控机制。已知OTUD5与PIF1存在有相互作用关系,OTUD5是否对PIF1发挥重要的作用尚不清楚,所以本课题将围绕OTUD5对PIF1的调控机制进行研究,进一步探索作为OUT家族重要成员OTUD5对PIF1的调控是否有重要的功能。方法:利用Western blot实验检测OTUD5过表达后内源及外源表达的PIF1蛋白的变化。采用RNA干扰技术敲低OTUD5检测外源表达的PIF1蛋白的变化。采用实时定量PCR(RT-PCR)检测OTUD5过表达或敲低对PIF1mRNA水平的影响,探讨OTUD5对PIF1转录水平的调节。利用半寿期实验检测OTUD5过表达后PIF1的降解速率,探讨OTUD5对PIF1蛋白稳定性的调节。运用泛素化实验检测OTUD5过表达或敲低对PIF1蛋白的泛素化的影响,探讨OTUD5对PIF1表达的调节作用。体外泛素化实验检测OTUD5过表达对PIF1全长蛋白及PIF1C截短体的去泛素化作用,判断PINT结构域在OTUD5去泛素化中的作用。结果:1.过表达OTUD5增加内源PIF1及外源表达PIF1蛋白含量。2.敲低OTUD5降低外源表达的PIF1蛋白含量。3.过表达OTUD5增加PIF1蛋白半寿期,但过表达或敲低OTUD5对PIF1 mRNA没有影响。4.过表达OTUD5降低PIF1蛋白的泛素化水平,敲低OTUD5增加了PIF1泛素化水平。5.PINT结构域缺失抑制了OTUD5对PIF1的去泛素化作用。结论:OTUD5通过与PIF1蛋白PINT结构域结合去泛素化PIF1增加PIF1蛋白稳定性。更多还原

【Abstract】 Objective: In this study,we investigated the mechanism of ubiquitination enzyme OTUD5 on PIF1 protein.It is known that OTUD5 interacts with PIF1.It is unclear whether OTUD5 plays an important role in PIF1.So this topic will focus on regulation mechanism of OTUD5 on PIF1 was studied.To further explore as OTU family members OTUD5 on regulation of PIF1 whether there is an important function.Methods: Westernblotwere used to detect the changes of endogenous and exogenous PIF1 protein after overexpression of OTUD5.RNA interference was used to knock down OTUD5 to detect the changes of exogenous expressed PIF1 protein.Real time quantitative PCR(RT-PCR)was used to detect the effect of over expression or knockdown of OTUD5 on the level of PIF1 mRNA.The degradation rate of after PIF1 overexpression of OTUD5 was detected by half-life assay,and the regulation of OTUD5 on the stability of PIF1 protein was investigated.Finally,ubiquitination testwas used to detect the overexpression or knockdown of OTUD5 on ubiquitination of PIF1 protein,and the regulation of OTUD5 on the expression of PIF1 was investigated.Finally,ubiquitination test in vitro was used to detect the over ubiquitination of PIF1 full-length protein and PIF1 C truncated by OTUD5,and to determine the role of PINT domain in ubiquitination of OTUD5.Results: 1.Overexpression of OTUD5 increased the content of endogenous PIF1 and exogenous expressed PIF1 protein.2.Knock down OTUD5 decreased the exogenous expression of PIF1 protein content.3.Overexpression of OTUD5 increased the half-life of PIF1 protein,but overexpression or knockdown of OTUD5 had no effect on PIF1 mRNA.4.Overexpression of OTUD5 reduced the ubiquitination level of PIF1 protein,and knockdown of OTUD5 increased the ubiquitination level of PIF1.5.Deletion of the PINT domain inhibits the ubiquitination of OTUD5 by PIF1.Conclusion: OTUD5 binds to the PIF1 protein PINT domain,and dubiquitination of PIF1 increases the stability of PIF1 proteins.更多还原