miRNA参与了生物体重要的基因表达调控过程,其中miR164通过对标靶NAC(NAM/ATAF/CUC)基因家族的精细调控,在植物激素信号传导、生长发育以及胁迫应答中起着重要作用。为了验证杨树中miR164a和其预测靶基因PeNAC1间是否也存在这一相互作用,笔者采用PCR技术克隆了毛果杨(Populus trichocarpa)miR164a的前体序列Ptc-MIR164a,并通过RNA fold(http://rna.tbi.univie.ac.at/)在线软件对其进行了miRNA二级结构分析。结果表明:该序列能形成典型的二级茎环结构,预示其在细胞内能被加工为成熟的miR164a。进一步借鉴动物细胞miRNA研究中荧光素酶报告基因法,利用高效的杨树原生质体瞬时表达体系,验证了Ptc-MIR164a对其预测靶基因PeNAC1的标靶作用; 同时,也建立了一种比较简单、直观的植物miRNA靶标基因的鉴定方法。
Abstract
miRNA plays a central role in gene regulations. Many research showed that miR164a targets NAC(NAM/ATAF/CUC)genes involved in multiple biological functions, such as plant development, hormones singal transduction and environmental stimuli. To confirm the interaction between micro164a and its predicted target gene PeNAC1 in poplar, the pre-micro164a sequence was cloned from P. trichocarpa genome DNA by PCR, named Ptc-MIR164a. And according to the results of secondary structure analyses with RNA fold(http://rna.tbi.univie.ac.at/), Ptc-MIR164a can form a typical stem-loop structure that suggests it can be processed into mature miRNA164a in vivo. Then combining the principle of luciferase reporter plasmids used in miRNA target detection in animal cell and the transient expression system of poplar protoplasts, we confirmed the interaction between Ptc-MIR164a and PeNAC1 and established a simple and intuitive method for miRNA target detection in plant cell.
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基金
收稿日期:2015-12-23 修回日期:2016-05-13
基金项目:国家林业公益性行业科研专项青年项目(20134111); 国家自然科学基金青年项目(31200507); 江苏高校优势学科建设工程资助项目(PAPD)
第一作者:王浩然(njlydxwhr@163.com)。*通信作者:陈英(ychen@ njfu.edu.cn),副教授。
引文格式:王浩然,李爽爽,乐丽娜,等. miR164a及其靶基因PeNAC1相互作用研究[J]. 南京林业大学学报(自然科学版),2016,40(5):29-33.
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