title: Plant viral leaders influence expression of a reporter gene in tobacco.
abstract: In order to optimise expression of a foreign protein in transgenic plants we investigated the potential benefits of including a viral untranslated leader sequence within a plant transformation vector. A variety of 5 leaders, including the tobacco mosaic virus (TMV) leader sequence and 31 nucleotides of the cauliflower mosaic virus (CaMV) 35S RNA leader, were compared. Viral leader constructs employing the 35S promoter and the reporter beta-glucuronidase (GUS) were tested by electroporation into tobacco mesophyll protoplasts and against a cointroduced chloramphenicol acetyl transferase (CAT) gene in transgenic tobacco leaves. In the transient assay system, GUS activities from the viral leaders were compared with those from either a short, random leader or a translational fusion of the CaMV 19S RNA ORF VI to GUS. A two- to-three-fold enhanced level of expression resulted when these leaders were substituted with either the 35S RNA or the TMV leader sequences. This enhancement was further increased, to four- to five-fold, by inclusion of four or seven of the bases from the 35S transcription initiation site adjacent to the TMV leader. In transgenic tobacco the improved GUS levels were maintained from constructs including either the TMV leader (eight-fold) or this sequence with the addition of the 35S transcription initiation site bases (ten-fold). A comparison of GUS enzyme amounts with GUS mRNA amounts, using the CAT gene as an internal standard, revealed that TMV leader-bearing mRNA was translated from four- to six-fold more efficiently than the random leader control.
authors.affiliation: AFRC-IPSR Nitrogen Fixation Laboratory, University of Sussex, Brighton, UK.
authors.forename: M J
authors.initials: MJ
authors.lastname: Dowson Day
authors.forename: J L
authors.initials: JL
authors.lastname: Ashurst
authors.forename: S F
authors.initials: SF
authors.lastname: Mathias
authors.forename: J W
authors.initials: JW
authors.lastname: Watts
authors.forename: T M
authors.initials: TM
authors.lastname: Wilson
authors.forename: R A
authors.initials: RA
authors.lastname: Dixon
mesh_terms: D001483:Base Sequence; D017796:Caulimovirus / Q000235:genetics*; D003001:Cloning, Molecular; D004279:DNA, Viral; D017930:Genes, Reporter; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism / Q000382:microbiology; D010947:Plants, Toxic; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics*; D015183:Restriction Mapping; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism / Q000382:microbiology; D014027:Tobacco Mosaic Virus / Q000235:genetics*; D014170:Transformation, Genetic
chemical_list: D004279:DNA, Viral; D012333:RNA, Messenger; D012367:RNA, Viral; D005966:Glucuronidase