Functional analysis of a Douglas-fir metallothionein-like gene promoter: transient assays in zygotic and somatic embryos and stable transformation in transgenic tobacco

• Published in: Planta 2004-11, Vol.220 (1), p.118-128
• Main Authors: Chatthai, M, Osusky, M, Osuka, L, Yevtushenko, D, Misra, S
• Format: Article
• Language: English
• Subjects: Agronomy. Soil science and plant productions; Amino Acid Sequence; amino acid sequences; Base Sequence; Biological and medical sciences; Complementary DNA; DNA, Plant - genetics; Embryos; Fundamental and applied biological sciences. Psychology; Genes; Genetic engineering applications; genetic transformation; Genetics and breeding of economic plants; Genomic Library; Genomics; Germination; metallothionein; Metallothionein - genetics; Molecular Sequence Data; Nicotiana - genetics; Nicotiana - metabolism; Nicotiana tabacum; nucleotide sequences; plant biochemistry; Plant breeding: fundamental aspects and methodology; plant genetics; Plant Proteins - genetics; Plants; Plants, Genetically Modified - metabolism; Promoter regions; Promoter Regions, Genetic - genetics; Proteins; Pseudotsuga - genetics; Pseudotsuga menziesii; recombinant fusion proteins; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Seedlings; Seeds; Somatic embryos; Tobacco; transgenes; Transgenic plants; zygote; Zygote - physiology
• ISSN: 0032-0935; 1432-2048
• DOI: 10.1007/s00425-004-1332-4

Douglas-fir (Pseudotsuga menziesii [Mirb] Franco) metallothionein (PmMT) cDNA encodes a novel cysteine- and serine-rich MT, indicating a new subtype or prototype MT from which other plant MTs may have evolved. A genomic library of Douglas-fir was screened using MT cDNA probes, and genomic sequences that mediate tissue-specific, temporal as well as inducible expression of the embryo-specific MT-gene were analyzed. The promoter region of the PmMT genomic clone (gPmMT) contained a hexameric G-box, two putative ethylene-responsive elements and an inverted repeat of a motif similar to the core metal regulatory element. Interestingly, comparison of the upstream region of Douglas-fir gPm2S1 and gPmMTa genes revealed a conserved motif, CATTATTGA, not found in any known angiosperm gene promoter. Chimeric gene constructs containing a series of deletions in the gPmMTa promoter fused to the uidA reporter gene were assayed in Douglas-fir and transgenic tobacco (Nicotiana tabacum L.). Transient-expression assays in Douglas-fir megagametophyte and zygotic embryos indicated that the sequence -190 to +88 of gPmMTa was sufficient to drive the expression of the reporter gene and that the 225-bp fragment (-677 to -453) contained sequences necessary for high-level expression. In transgenic tobacco seedlings the beta-glucuronidase activity was localized in the vacuolar tissue and proliferating tissue of the auxiliary buds and stem elongation zone. The gPmMTa promoter was not active in the seeds of transgenic tobacco or in the roots of seedlings up to 3 weeks old. Detailed studies of transient expression and stable transformation provided important information on evolutionary conservation as well as novel features found in the conifer promoter. This is the first report of an MT-like gene promoter from conifers.