Ju Zheng, Cao Dongyan, Gao Chao, Zuo Jinhua, Zhai Baiqiang, Li Shan, Zhu Hongliang, Fu Daqi, Luo Yunbo, Zhu Benzhong
College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China (Z.J., D.C., Ba.Z., S.L., H.Z., D.F., Y.L., Be.Z.).
Biotechnology Research Center, Shandong Academy of Agricultural Sciences, Jinan 250100, China (C.G.); and.
Plant Physiol. 2017 Apr;173(4):1940-1952. doi: 10.1104/pp.16.01489. Epub 2017 Feb 22.
With experimental and bioinformatical methods, numerous small RNAs, including microRNAs (miRNAs) and short interfering RNAs (siRNAs), have been found in plants, and they play vital roles in various biological regulation processes. However, most of these small RNAs remain to be functionally characterized. Until now, only several viral vectors were developed to overexpress miRNAs with limited application in plants. In this study, we report a new small RNA overexpression system via viral satellite DNA associated with (TYLCCNV) vector, which could highly overexpress not only artificial and endogenous miRNAs but also endogenous siRNAs in First, we constructed basic TYLCCNV-amiRPDS(319L) vector with widely used backbone, but the expected photobleaching phenotype was very weak. Second, through comparing the effect of backbones (, , and ) on specificity and significance of generating small RNAs, the backbone was optimally selected to construct the small RNA overexpression system. Third, through sRNA-Seq and Degradome-Seq, the small RNAs from backbone in TYLCCNV-amiRPDS(390) vector were confirmed to highly overexpress amiRPDS and specifically silence targeted gene. Using this system, rapid functional analysis of endogenous miRNAs and siRNAs was carried out, including miR156 and 5'D8(+). Meanwhile, through designing corresponding artificial miRNAs, this system could also significantly silence targeted endogenous genes and show specific phenotypes, including , , and These results demonstrated that this small RNA overexpression system could contribute to investigating not only the function of endogenous small RNAs, but also the functional genes in plants.
通过实验和生物信息学方法，在植物中发现了众多小RNA，包括微小RNA（miRNA）和小干扰RNA（siRNA），它们在各种生物调控过程中发挥着至关重要的作用。然而，这些小RNA中的大多数仍有待进行功能表征。到目前为止，仅开发了几种病毒载体来在植物中过表达miRNA，但其应用有限。在本研究中，我们报道了一种与番茄黄化曲叶中国病毒（TYLCCNV）载体相关的新型小RNA过表达系统，该系统不仅可以在本氏烟草中高效过表达人工和内源miRNA，还能过表达内源siRNA。首先，我们构建了具有广泛使用的骨架的基本TYLCCNV-amiRPDS(319L)载体，但预期的光漂白表型非常弱。其次，通过比较骨架（pBinPLUS、pCAMBIA1300和pGreenII 0029）对产生小RNA的特异性和重要性的影响，最佳选择了pGreenII 0029骨架来构建小RNA过表达系统。第三，通过sRNA-Seq和降解组测序（Degradome-Seq）证实，TYLCCNV-amiRPDS(390)载体中pGreenII 0029骨架产生的小RNA能高效过表达amiRPDS并特异性沉默靶向的PDS基因。利用该系统对内源miRNA和siRNA进行了快速功能分析，包括miR156和5'D8(+)。同时，通过设计相应的人工miRNA，该系统还能显著沉默靶向的内源基因并表现出特定表型，包括PDS、AGO1和NRPD1。这些结果表明，这种小RNA过表达系统不仅有助于研究内源小RNA的功能，还有助于研究植物中的功能基因。
