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Position: Home > Articles > A Novel Method for Constructing Small RNA Expression Vector——Artificial pre-MircoRNA Biotechnology Bulletin 2010 (7) 101-105

小分子RNA表达载体构建的新方法——MicroRNA前体PCR置换法

作  者：
陈锐;胡正;张辉
单  位：
中国农业科学院作物科学研究所
关键词：
小分子RNA;MicroRNA;PCR置换法
摘  要：
MicroRNA(miRNA)基因的终产物是进化上高度保守的、具有基因表达调控功能的非编码小分子RNA。miRNA的特征性发卡环前体(pre-miRNAs)在体内经数步加工后,与AGO蛋白构成沉默复合体(RISC)以行使其功能。如将已知pre-miRNAs上的miRNA成熟链和互补链(miRNA*)分别置换为有待研究的小分子RNA,并构建表达载体转化植株,利用转化株内源的miRNA成熟加工体系,可以产生预设的小分子RNA。利用已知拟南芥miRNA前体为模板,使用替换PCR的方法,人工构建了gso8-ap-miRNA-pBI121表达载体。利用农杆菌介导法转化拟南芥,多数T1代植株表现出提早开花的突变表型。该方法是一种简便、高效的构建小分子RNA表达载体的方法。
译  名：
A Novel Method for Constructing Small RNA Expression Vector——Artificial pre-MircoRNA
作  者：
Chen Rui Hu Zheng Zhang Hui ( Crop Sciences Institute,Chinese Academy of Agricultural Sciences,National Key Facility for Crop Gene Resources and Genetic Improvement,Beijing 100081)
关键词：
Small RNA MicroRNA Over-lap PCR
摘  要：
The end products of MicroRNA genes( MIRs) are small non-coding RNAs,which are part of an evolutionarily highly conserved intracellular mechanism to regulate gene expression in a sequence-specific manner. Mature miRNAs were generated from miRNA precursors( pre-miRNAs) with characteristic hairpin structures by sequential processing steps before they were loaded into the RISC complex for exerting functions. If we replace the miRNA and miRNA of the pre-miRNA and construct expression vector for transformation,the artificial pre-miRNA would be processed by endogenous mechanisms and generate pre-designed small RNA after transformation. Here,we used the known Arabidopsis pre-miRNA as template to preform replacement successfully by over-lap PCR method and constructed gso8-ap-miRNA-pBI121 expression vector. After Agrobacterium-mediated transformation of Arabidopsis thaliana,most transgenic plantlets exhibited early flowering mutant phenotype. This method was proved to be a simple and efficient approach for small RNA expression vector construction by PCR detection.

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