《植物生理学报》 2010, 46(7): 693-700

人工microRNA干扰拟南芥AtCDKC;1 和AtCDKC;2 基因表达的初步研究

赵臻, 邱凯, 蒯本科*

复旦大学生命科学学院, 遗传工程国家重点实验室和植物科学研究所, 上海200433

通信作者：蒯本科；E-mail: bkkuai@fudan.edu.cn；Tel: 021-65642648

摘　要：

以来源于拟南芥的 microRNA 序列为骨架, 构建抑制 AtCDKC;1 和 AtCDKC;2 基因的人工 microRNAs 载体, 研究其对 目的基因表达的抑制效果。选择 AtCDKCs 基因的特异性序列, 通过重叠 PCR 的方法改造拟南芥 microRNA164a 骨架序列, 连接到双元载体 pPZPY122, 在农杆菌介导下转化拟南芥。RT-PCR 分析表明, 人工microRNA 能够显著抑制目的基因的表 达, 获得了抑制效果明显的转基因植株, 并且对AtCDKCs在拟南芥生长发育中的作用进行了初步的研究。

关键词：周期蛋白依赖性激酶; 人工microRNA; RNA干扰; 基因表达; 拟南芥发育

收稿：2010-04-20   修定：2010-05-14

A Preliminary Analysis of Artificial MicroRNAs-Mediated Interference of AtCDKC;1 and AtCDKC;2 Expression in Arabidopsis

ZHAO Zhen, QIU Kai, KUAI Ben-Ke*

State Key Laboratory of Genetic Engineering and Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai 200433, China

Corresponding author: KUAI Ben-Ke; E-mail: bkkuai@fudan.edu.cn; Tel: 021-65642648

Abstract:

By using Arabidopsis endogenous microRNA sequences, artificial microRNAs (amiRNAs) were generated to knock down the expressions of AtCDKCs. To construct plasmids, oligonucleotide sequences targeting AtCDKCs-specific locus were used to engineer microRNA164a by overlapping PCR and the resultant sequence was inserted into the binary vector pPZPY122. Arabidopsis of wild type (Col-0) was transformed by the Agrobacterium-mediated approach. RT-PCR analysis showed that the target genes were both specifically and efficiently silenced in transgenic lines. The roles of AtCDKCs in the growth and development of Arabidopsis was analyzed based on the transgenic plants.

Key words: AtCDKC; artificial microRNA; RNA interference; gene expression; Arabidopsis development