title: A Viral Satellite DNA Vector (TYLCCNV) for Functional Analysis of miRNAs and siRNAs in Plants.
abstract: 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 withTomato yellow leaf curl China virus(TYLCCNV) vector, which could highly overexpress not only artificial and endogenous miRNAs but also endogenous siRNAs inNicotiana benthamianaFirst, we constructed basic TYLCCNV-amiRPDS(319L) vector with widely usedAtMIR319abackbone, but the expected photobleaching phenotype was very weak. Second, through comparing the effect of backbones (AtMIR319a,AtMIR390a, andSlMIR159) on specificity and significance of generating small RNAs, theAtMIR390abackbone was optimally selected to construct the small RNA overexpression system. Third, through sRNA-Seq and Degradome-Seq, the small RNAs fromAtMIR390abackbone in TYLCCNV-amiRPDS(390) vector were confirmed to highly overexpress amiRPDS and specifically silence targetedPDSgene. Using this system, rapid functional analysis of endogenous miRNAs and siRNAs was carried out, including miR156 andathTAS3a5'D8(+). Meanwhile, through designing corresponding artificial miRNAs, this system could also significantly silence targeted endogenous genes and show specific phenotypes, includingPDS,Su, andPCNAThese 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.
authors.affiliation: 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.|National Engineering Research Center for Vegetables, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China (J.Z.).
authors.forename: Zheng
authors.initials: Z
authors.lastname: Ju
authors.affiliation: 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.|National Engineering Research Center for Vegetables, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China (J.Z.).
authors.forename: Dongyan
authors.initials: D
authors.lastname: Cao
authors.affiliation: 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.|National Engineering Research Center for Vegetables, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China (J.Z.).
authors.forename: Chao
authors.initials: C
authors.lastname: Gao
authors.affiliation: 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.|National Engineering Research Center for Vegetables, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China (J.Z.).
authors.forename: Jinhua
authors.initials: J
authors.lastname: Zuo
authors.affiliation: 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.|National Engineering Research Center for Vegetables, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China (J.Z.).
authors.forename: Baiqiang
authors.initials: B
authors.lastname: Zhai
authors.affiliation: 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.|National Engineering Research Center for Vegetables, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China (J.Z.).
authors.forename: Shan
authors.initials: S
authors.lastname: Li
authors.affiliation: 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.|National Engineering Research Center for Vegetables, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China (J.Z.).
authors.forename: Hongliang
authors.identifier: 0000-0002-1484-2212
authors.initials: H
authors.lastname: Zhu
authors.affiliation: 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.|National Engineering Research Center for Vegetables, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China (J.Z.).
authors.forename: Daqi
authors.initials: D
authors.lastname: Fu
authors.affiliation: 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.|National Engineering Research Center for Vegetables, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China (J.Z.).
authors.forename: Yunbo
authors.initials: Y
authors.lastname: Luo
authors.affiliation: 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.); zbz@cau.edu.cn.|Biotechnology Research Center, Shandong Academy of Agricultural Sciences, Jinan 250100, China (C.G.); and zbz@cau.edu.cn.|National Engineering Research Center for Vegetables, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China (J.Z.) zbz@cau.edu.cn.
authors.forename: Benzhong
authors.identifier: 0000-0002-7089-4703
authors.initials: B
authors.lastname: Zhu
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000821:virology; D001483:Base Sequence; D053839:Begomovirus / Q000235:genetics* / Q000502:physiology; D004276:DNA, Satellite / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics*; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D018551:Solanum lycopersicum / Q000821:virology; D035683:MicroRNAs / Q000235:genetics*; D018515:Plant Leaves / Q000235:genetics / Q000821:virology; D010944:Plants / Q000235:genetics* / Q000821:virology; D034741:RNA, Small Interfering / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017423:Sequence Analysis, RNA / Q000379:methods; D012689:Sequence Homology, Nucleic Acid; D014026:Nicotiana / Q000235:genetics / Q000821:virology
chemical_list: D004276:DNA, Satellite; D004279:DNA, Viral; C584086:MIRN390 microRNA, Arabidopsis; D035683:MicroRNAs; D034741:RNA, Small Interfering