拟南芥At5g01510和At5g49820基因人工microRNAs的构建与鉴定

郭凯华, 李文超, 李思敏, 赵淑清*
山西大学生物技术研究所, 化学生物学与分子工程教育部重点实验室, 太原030006

通信作者:赵淑清;E-mail: shuqin@sxu.edu.cn;Tel: 0351-7016123

摘 要:

以拟南芥内源MIR319a前体为骨架, 构建沉默DUF647家族基因At5t01510和At5g49820表达的人工microRNAs, 研究其 对目的基因表达的抑制效果。利用WMD平台设计分别靶向At5g01510和At5g49820的amiRNAs序列, 通过重叠PCR改造拟 南芥MIR319a骨架序列, 使其包含我们设计的特异amiRNAs序列。构建35S::amiR-At5g0150和35S::amiR-At5g49820融合基 因, 以农杆菌介导的花苞浸染法转化获得转基因拟南芥。RT-PCR分析表明, 人工microRNAs能够显著抑制靶基因的表达, 获得了抑制效果明显的转基因植株。本工作为进一步研究这两个基因的功能奠定了良好的基础。

关键词:拟南芥; At5g01510; At5g49820; 人工microRNAs; 基因表达

收稿:2011-11-29   修定:2012-03-28

资助:国家自然科学基金(31170273)、山西省国际科技合作计 划( 2 0 0 9 0 8 1 0 0 5 ) 、山西省回国留学人员科研资助项目 (201003)、山西省留学人员科技活动项目择优资助和太原 市科技明星专项(11014902)。

Construction and Identification of Artificial MicroRNAs Targeting Arabidopsis At5g01510 and At5g49820 Genes

GUO Kai-Hua, LI Wen-Chao, LI Si-Min, ZHAO Shu-Qing*
Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan 030006, China

Corresponding author: ZHAO Shu-Qing; E-mail: shuqin@sxu.edu.cn; Tel: 0351-7016123

Abstract:

Using the endogenous Arabidopsis MIR319a precursor as a backbone, we constructed two artificial microRNAs (amiRNAs) to knock down the expressions of At5g01510 and At5g49820 genes. By using the WMD (Web MicroRNA Designer) platform, we designed two amiRNAs targeting the genes At5g01510 and At5g49820, respectively. Both amiRNAs were engineered into the MIR319a precursor using overlapping PCR to replace the endogenous miRNA sequences, and the amiRNAs backbones were fused to the constitutive promoter within the binary vector pCHF3. The amiR-At5g01510 and amiR-At5g49820 expression constructs were introduced into Arabidopsis wild type (Col-0) by Agrobacterium-mediated floral dipping. RT-PCR analysis showed that the target genes were efficiently knocked down in transgenic lines harboring the overexpression constructs. This work has laid a foundation for further study of the functions of At5g01510 and At5g49820 genes.

Key words: Arabidopsis thaliana; At5g01510; At5g49820; amiRNAs; gene expression

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