title: Consistent transcriptional silencing of 35S-driven transgenes in gentian.
abstract: In this study, no transgenic gentian (Gentiana triflora x Gentiana scabra) plants produced via Agrobacterium-mediated transformation exhibited transgene (GtMADS, gentian-derived MADS-box genes or sGFP, green fluorescent protein) expression in their leaf tissues, despite the use of constitutive Cauliflower mosaic virus (CaMV) 35S promoter. Strikingly, no expression of the selectable marker gene (bar) used for bialaphos selection was observed. To investigate the possible cause of this drastic transgene silencing, methylation-specific sequences were analysed by bisulfite genomic sequencing using tobacco transformants as a control. Highly methylated cytosine residues of CpG and CpWpG (W contains A or T) sites were distinctively detected in the promoter and 5' coding regions of the transgenes 35S-bar and 35S-GtMADS in all gentian lines analysed. These lines also exhibited various degrees of cytosine methylation in asymmetrical sequences. The methylation frequencies in the other transgene, nopaline synthase (NOS) promoter-driven nptII, and the endogenous GtMADS gene coding region, were much lower and were variable compared with those in the 35S promoter regions. Transgene methylation was observed in the bialaphos-selected transgenic calluses expressing the transgenes, and methylation sequences were distributed preferentially around the as-1 element in the 35S promoter. Calluses derived from leaf tissues of silenced transgenic gentian also exhibited transgene suppression, but expression was recovered by treatment with the methylation inhibitor 5-aza-2'-deoxycytidine (aza-dC). These results indicated that cytosine methylation occurs exclusively in the 35S promoter regions of the expressed transgenes during selection of gentian transformants, causing transcriptional gene silencing.
authors.affiliation: Iwate Biotechnology Research Center, 22-174-4 Narita, Kitakami, Iwate 024-0003, Japan. mishiba@plant.osakafu-u.ac.jp
authors.forename: Kei-ichiro
authors.initials: K
authors.lastname: Mishiba
authors.forename: Masahiro
authors.initials: M
authors.lastname: Nishihara
authors.forename: Takashi
authors.initials: T
authors.lastname: Nakatsuka
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Abe
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Hirano
authors.forename: Takahide
authors.initials: T
authors.lastname: Yokoi
authors.forename: Akiko
authors.initials: A
authors.lastname: Kikuchi
authors.forename: Saburo
authors.initials: S
authors.lastname: Yamamura
mesh_terms: D001483:Base Sequence; D017796:Caulimovirus / Q000235:genetics; D019175:DNA Methylation; D004269:DNA, Bacterial / Q000235:genetics; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics; D004274:DNA, Recombinant / Q000737:chemistry / Q000235:genetics; D020868:Gene Silencing; D030012:Gentiana / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D026161:MADS Domain Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics; D012231:Rhizobium / Q000235:genetics; D014026:Nicotiana / Q000235:genetics; D014170:Transformation, Genetic
chemical_list: D004269:DNA, Bacterial; D018744:DNA, Plant; D004274:DNA, Recombinant; D026161:MADS Domain Proteins; D010940:Plant Proteins; D011994:Recombinant Proteins; C036483:T-DNA; D049452:Green Fluorescent Proteins