Construction of Multi-ribozyme Expression System and Its Characterization of Cleavage on the MDR1/MRP1 Double Target Substrate in vitro

To improve catalytic activity of ribozyme on its substrate,the multi-ribozyme expression system was designed and constructed from 20 cis-acting hammerhead ribozymes undergoing self-cleavage with 10 trans-acting hammerhead ribozymes inserted altematively regularly and the plasmid of pGEM-MDRI/MRPI used to transcribe the M DRI/MRPI(196/210) substrate containing double target sites was also constructed by DNA recombination.Endonuclease digestion analysis and DNA sequencing indicate all the recombinant plasmids were correct.The cleavage activities were evaluated for the multi-ribozyme expression system on the MDR1/MRP1 substrate in the cell free system.The results demonstrate that the cis-acting hammerhead ribozymes in the multi-ribozyme expression system were able to cleave themselves and the 72 nt of 196Rz and the 71 nt of 210Rz trans-acting hammerhead ribozymes were liberated effectively,and the trans-acting hammerhead ribozymes released were able to act on the MDR1/MRP1 double target RNA substrate and cleave the target RNA at specific sites effectively.The multiribozyme expression system of the [Coat'A196Rz/Coat'B210Rz]5 is more significantly superior to that of the [Coat'A 196Rz/Coat'B210Rz]1 in cleavage of RNA substrate.The fractions cleaved by [Coat'A196Rz/Coat'B210Rz]5 on the MDR1/MRP1 substrate for 8 h at observed temperatures showed no marked difference.The studies of Mg2+ on cleavage efficiency indicate that cleavage reaction is dependent on Mg2+ ions concentration.The plot of Ig(kobs) vs.Igc(Mg2+) displays a linear relationship between 2.5 mmol/L and 20 mmol/L Mg2..It suggests that Mg2+ ions play a crucial role in multi-ribozyme cleavage on the substrate.     

排列游戏问题及其数学表达

利用排列逆序数定理讨论两个排列游戏问题,否定了其操作的可行性．并就其中一个问题作了一般性研究,给出了该类型游戏是否可行的充要条件,更进一步得出了完成该类游戏的最少操作次数及其变式问题的可行性操作次数．在此基础上,导出一个关于矩阵的命题．     

基于数学核心素养提升的“阅读—思考—表达”学习模式构建及案例开发

《普通高中数学课程标准(2017年版2020年修订)》在课程目标中对学生的阅读能力、思考能力与表达能力提出了相应的要求.本文构建了“阅读—思考—表达”的学习模式,以促进学生阅读力、思考力、表达力的全面提升,是对学生数学核心素养落地与培育的一次全新探索.在探讨“阅读—思考—表达”学习模式的内涵和价值之后,构建了基于“阅读—思考—表达”学习模式对应的教学模式框架,并开发设计了典型案例“数列的通项公式”.