Summary
A chimeric tubulin gene has been constructed by the fusion of a genomic sequence containing a truncated soybean beta-tubulin gene with 2 kb of upstream DNA to the 3′ untranslated region containing the polyadenylation signal from transcription unit 7 of the octopine Ti plasmid pGV117. The chimeric gene has been incorporated into the Ti plasmid transformation vector pGV3850::pAP2034, along with a selectable marker gene active in plants (the neomycin phosphotransferase II gene, conferring kanamycin resistance). Strains of Agrobacterium tumefaciens haboring the plasmids were used to transform Nicotiana tabacum by the leaf disk method and plants were regenerated from transformed cells. Transgenic plants were self fertilized and the segregation of kanamycin resistance was assayed. DNA and RNA were extracted from transgenic plants, fractionated by agarose gel electrophoresis, blotted to nitrocellulose and hybridized to a probe specific for the chimeric gene to assess its structure and expression in transgenic plants. The chimeric gene was stably integrated into the tobacco genome without rearrangements and it was expressed as a polyadenylated RNA of 1.7 kb in the transformants. Genetic analysis revealed that the kanamycin-resistant phenotype was inherited in a Mendelian fashion over two generations.
Similar content being viewed by others
References
Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523
Burland TG, Gull K, Schedl T, Boston RS, Dove WF (1983) Cell type-dependent expression of tubulins in Physarum. J Cell Biol 97:1852–1859
Bustos MM, Guiltinan MJ, Cyr RJ, Fosket DE (1986) Developmental regulation of tubulin mRNA in soybean. Planta
Cashmore AR (1982) The isolation of poly(A)+ messenger RNA from plants. In: Edelman M, Hallick RB, Chua N-H (eds) Methods in Chloroplast Molecular Biology. Elsevier Biomedical Press, New York, p 387
Chen EY, Seeburgh EH (1985) Supercoil sequencing: A fast and simple method for sequencing plasmid DNA. DNA 4:165–170
Cleveland DW, Sullivan KF (1985) Molecular biology and genetics of tubulin. Annu Rev Biochem 54:331–365
Cowan NJ, Dobner PR, Fuchs EV, Cleveland DW (1983) Expression of human alpha-tubulin genes: Interspecies conservation of 3′ untranslated regions. Mol Cell Biol 3:1738–1745
Cyr RJ, Bustos MM, Guiltinan MJ, Fosket DE (1986) Developmental modulation of tubulin protein and mRNA levels during carrot (Daucus carota L) somatic embryogenesis. Planta
Dhaese P, De Greve H, Decraemer H, Schell J, Van Montagu M (1979) Rapid mapping of transposon insertion and deletion mutations in the large plasmids of Agrobacterium tumefaciens. Nucleic Acids Res 7:1837–1849
Dhaese P, De Greve H, Gielen J, Seurinck J, Van Montagu M, Schell J (1983) Identification of sequences involved in the polyadenylation of higher plant nuclear transcripts using Agrobacterium tumefaciens. EMBO J 2:419–426
Green MR, Maniatis T, Melton DA (1983) Human beta-globin pre-mRNA synthesized in vitro is accurately spliced in Xenopus oocyte nuclei. Cell 32:681–694
Guiltinan MJ, Bustos MM, Cyr RJ, Fosket DE (1986) Isolation and characterization of beta-tubulin genes from soybean (Glycine max L.). Plant Mol Biol
Hanahan D (1983) Studies on transformations of Escherichia coli with plasmids. J Mol Biol 166:557–580
Horsch RB, Fry JE, Hoffman NL, Eichholtz D, Rogers SG, Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227:1229–1231
Kirschner MW (1978) Microtubule assembly and nucleation. Int Rev Cytol 54:1–71
Krauhs E, Little M, Kempf T, Hofer-Warbinek R, Ade W, Ponstingl H (1981) complete amino acid sequence of beta-tubulin from porcine brain. Proc Natl Acad Sci USA 78:5156–5160
Lehrach H, Diamond D, Wozney JM, Boedtker H (1977) RNA molecular weight determinations by gel electrophoresis under denaturing conditions. Biochemistry 16:4743–4751
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular Cloning, a Laboratory Manual. Cold Spring Harbor Laboratory, New York
Melton D (1985) Injected anti-sense RNAs specifically block messenger RNA translation in vivo. Proc Natl Acad Sci USA 82:144–148
Melton D, Krieg PA, Rebagliati MR, Maniatis T, Zinn K, Green MR (1984) Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res 12:7035–7056
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with two tobacco tissue cultures. Physiol Plant 15:473–497
Poncz M, Solowiejczyk D, Balantine M, Schwartz E, Surrey S (1982) “Nonrandom” DNA sequence analysis in bacteriophage M13 by the dideoxy chain-termination method. Proc Natl Acad Sci USA 79:4298–4302
Ponstingle H, Krauhs E, Little M, Kempf K (1981) Complete amino acid sequence of alpha-tubulin from porcine brain. Proc Natl Acad Sci USA 78:2757–2761
Sanger F, Coulson AR (1975) A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. J Mol Biol 94:441–448
Sanger F, Coulson AR (1978) Use of thin acrylamide gels for DNA sequencing. FEBS Lett 87:107–110
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Schell J, Van Motagu M, Holsters M, Zambryski P, Joos H, Herrera-Estrella L, Depicker A, Hernalsteens JP, De Greve H, Willmitzer L, Schröder J (1984) Ti plasmids as gene vectors for plants. In: Kumar A (ed) Eukaryotic Gene Expression. Plenum Press, New York, pp 141–160
Taylor B, Powell A (1983) Isolation of plant DNA and RNA. Focus (BRL) 4:4–6
Thomas TL, Hall TC (1985) Gene transfer and expression in plants: Implications and potential. Bioessays 3:149–153
Van Haute E, Joos H, Maes M, Warren G, Van Montagu M, Schell J (1983) Intergeneric transfer and exchange recombination of restriction fragments cloned in pBR 322: a novel strategy for reversed genetics of the Ti plasmids of Agrobacterium tumefaciens. EMBO J 2:411–417
Velten J, Schell J (1985) Selection-expression plasmid vectors for use in genetic transformation of higher plants. Nucleic Acids Res 13:6981–6998
Velten J, Velten L, Hain R, Schell J (1984) Isolation of a dual plant promoter fragment from the Ti plasmid of Agrobacterium tumefaciens. EMBO J 3:2723–2730
Weatherbee JA, May GS, Gambling J, Morris NR (1985) Involvement of a particular species of beta-tubulin (beta3) in conidial development in Aspergillus nidulans. J Cell Biol 101:706–711
Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequence of the M13mp18 and pUC19 vectors. Gene 33:103–119
Zambryski P, Joos H, Genetello C, Leemans J, Van Montagu M, Schell J (1983) Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity. EMBO J 2:2143–2150
Author information
Authors and Affiliations
Additional information
Communicated by R. Goldberg
Rights and permissions
About this article
Cite this article
Guiltinan, M.J., Velten, J., Bustos, M.M. et al. The expression of a chimeric soybean beta-tubulin gene in tobacco. Mol Gen Genet 207, 328–334 (1987). https://doi.org/10.1007/BF00331597
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00331597