人工miRNA沉默基因研析.pdfVIP

生物技术通报 ·技术与方法· BIOTECHNOLOGY BULLETIN 年第 期 2014 4 人工 miRNA 沉默基因的研究 张献贺  孔稳稳  李勇  李晶 （东北农业大学生命利学学院 农业生物功能基因重点实验室，哈尔滨 150030） 摘 要 ： 人工miRNA （amiRNA ）是一种高效的基因沉默技术，其原理是以植物内源miRNA 的前体为基本骨架，将其茎环 结构中的miRNA/miRNA* 序列替换为amiRNA/amiRNA* 序列，使产生的amiRNA 作用于目标靶基因，以实现对靶基因表达的抑制。 以拟南芥中转录因子基因MYB28 为例，介绍了amiRNA 的设计基本原理和构建方法。在MYB28 基因序列的3端、5端和中间部 分分别设计了amiRNA ，以拟南芥内源的miRNA 319a 前体为骨架，以靶向于MYB28 的amiRNA 序列替代原有的miRNA 319a 序列， 在拟南芥中过量表达，获得的转基因植株中MYB28 表达水平平均降低了86%。根据试验结果对人工miRNA 沉默基因技术的特点 及应用前景进行了讨论。 关键词 ： 人工miRNA RNA 干扰 拟南芥 基因沉默 Study of Gene Silencing by Artificial miRNA Zhang Xianhe Kong Wenwen Li Yong Li Jing （Key Laboratory of Agriculatural Biological Functional Genes ，College of Lif e Science ，Northeast Agricultural University ，Harbin 150030 ） Abstract:  Artificial miRNA （amiRNA ）is an effective gene silencing technology. The basic principle of amiRNA is based on its biogenesis mechanism. Using endogenous miRNA precursor as a basic framework, miRNA/miRNA* is then replaced by amiRNA/amiRNA*. The produced amiRNA is expected to silence the target gene. In this paper, the basic principle and method of amiRNA construction was introduced. AmiRNAs were designed to the target gene MYB28 as an example. Three amiRNAs respectively target to the 3 terminal region, 5 region and middle region of MYB28 coding sequence were designed. Precursor of miRNA 319a in Arabidopsis thaliana was taken as a framework and miRNA319a sequence was replaced by amiRNAs targeting to MYB28 gene. The expression level of MYB28 in transgenic plants decreased 86%. Based on our exper