Teotia Sachin, Tang Guiliang
College of Agronomy, Henan Agricultural University, Zhengzhou, 450002, China.
School of Biotechnology, Gautam Buddha University, Greater Noida, 201312, India.
Methods Mol Biol. 2017;1631:337-348. doi: 10.1007/978-1-4939-7136-7_22.
In plants, microRNAs (miRNAs) regulate more than hundred target genes comprising largely transcription factors that control growth and development as well as stress responses. However, the exact functions of miRNA families could not be deciphered because each miRNA family has multiple loci in the genome, thus are functionally redundant. Therefore, an ideal approach to study the function of a miRNA family is to silence the expression of all members simultaneously, which is a daunting task. However, this can be partly overcome by Target Mimic (TM) approach that can knockdown an entire miRNA family. STTM is a modification of TM approach and complements it. STTMs have been successfully used in monocots and dicots to block miRNA functions. miR159 has been shown to be differentially regulated by various abiotic stresses including ABA in various plant species. Here, we describe in detail the protocol for designing STTM construct to block miR159 functions in Arabidopsis, with the potential to apply this technique on a number of other stress-regulated miRNAs in plants.
在植物中，微小RNA（miRNA）调控着一百多个靶基因，这些靶基因主要包括控制生长发育以及应激反应的转录因子。然而，由于每个miRNA家族在基因组中都有多个位点，功能上存在冗余，因此无法确切解析miRNA家族的具体功能。所以，研究miRNA家族功能的理想方法是同时沉默所有成员的表达，但这是一项艰巨的任务。不过，通过靶标模拟（TM）方法可以部分克服这一难题，该方法能够敲低整个miRNA家族。短串联靶标模拟（STTM）是TM方法的一种改进，对其起到补充作用。STTM已成功应用于单子叶植物和双子叶植物，以阻断miRNA的功能。在多种植物物种中，miR159已被证明受包括脱落酸（ABA）在内的各种非生物胁迫的差异调控。在此，我们详细描述了设计STTM构建体以阻断拟南芥中miR159功能的方案，该技术有可能应用于植物中其他一些受胁迫调控的miRNA。
