Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1989 Nov;86(21):8467–8471. doi: 10.1073/pnas.86.21.8467

High-frequency T-DNA-mediated gene tagging in plants.

C Koncz 1, N Martini 1, R Mayerhofer 1, Z Koncz-Kalman 1, H Körber 1, G P Redei 1, J Schell 1
PMCID: PMC298303  PMID: 2554318

Abstract

An insertion element [transferred DNA (T-DNA)], transferred by soil agrobacteria into the nuclear genome of plants, was used for induction of gene fusions in Arabidopsis thaliana, Nicotiana tabacum, and Nicotiana plumbaginifolia. A promoterless aph(3')II (aminoglycoside phosphotransferase II) reporter gene was linked to the right end of the T-DNA and transformed into plants along with a plasmid replicon and a selectable hygromycin-resistance gene. Transcriptional and translational reporter gene fusions were identified by screening for APH(3')II enzyme activity in diverse tissues of transgenic plants. The frequency of gene fusions, estimated by determination of the copy number of T-DNA insertions, showed that on average 30% of T-DNA inserts induced gene fusions in Arabidopsis and Nicotiana. Gene fusions were rescued from plants by transformation of the T-DNA-linked plasmid and flanking plant DNA into Escherichia coli. By dissection of gene fusions and construction of chimeric genes, callus- and root-specific promoters were identified that showed an altered tissue specificity in the presence of a 3'-downstream-located 35S promoter. Transcript mapping of a gene fusion and expression of a non-frame transcriptional fusion of bacterial luciferase luxA and luxB genes demonstrated that dicistronic transcripts are translated in tobacco.

Full text

PDF
8467

Images in this article

8470Image
on p.8470
8470Image
on p.8470
8471Image
on p.8471

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baldwin T. O., Berends T., Bunch T. A., Holzman T. F., Rausch S. K., Shamansky L., Treat M. L., Ziegler M. M. Cloning of the luciferase structural genes from Vibrio harveyi and expression of bioluminescence in Escherichia coli. Biochemistry. 1984 Jul 31;23(16):3663–3667. doi: 10.1021/bi00311a014. [DOI] [PubMed] [Google Scholar]
  2. Beck E., Ludwig G., Auerswald E. A., Reiss B., Schaller H. Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5. Gene. 1982 Oct;19(3):327–336. doi: 10.1016/0378-1119(82)90023-3. [DOI] [PubMed] [Google Scholar]
  3. Dean C., Favreau M., Dunsmuir P., Bedbrook J. Confirmation of the relative expression levels of the Petunia (Mitchell) rbcS genes. Nucleic Acids Res. 1987 Jun 11;15(11):4655–4668. doi: 10.1093/nar/15.11.4655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Feldmann K. A., Marks M. D., Christianson M. L., Quatrano R. S. A Dwarf Mutant of Arabidopsis Generated by T-DNA Insertion Mutagenesis. Science. 1989 Mar 10;243(4896):1351–1354. doi: 10.1126/science.243.4896.1351. [DOI] [PubMed] [Google Scholar]
  5. Goldberg R. B., Hoschek G., Kamalay J. C., Timberlake W. E. Sequence complexity of nuclear and polysomal RNA in leaves of the tobacco plant. Cell. 1978 May;14(1):123–131. doi: 10.1016/0092-8674(78)90307-0. [DOI] [PubMed] [Google Scholar]
  6. Kamalay J. C., Goldberg R. B. Regulation of structural gene expression in tobacco. Cell. 1980 Apr;19(4):935–946. doi: 10.1016/0092-8674(80)90085-9. [DOI] [PubMed] [Google Scholar]
  7. Koncz C., Olsson O., Langridge W. H., Schell J., Szalay A. A. Expression and assembly of functional bacterial luciferase in plants. Proc Natl Acad Sci U S A. 1987 Jan;84(1):131–135. doi: 10.1073/pnas.84.1.131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  9. Olsson O., Escher A., Sandberg G., Schell J., Koncz C., Szalay A. A. Engineering of monomeric bacterial luciferases by fusion of luxA and luxB genes in Vibrio harveyi. Gene. 1989 Sep 30;81(2):335–347. doi: 10.1016/0378-1119(89)90194-7. [DOI] [PubMed] [Google Scholar]
  10. Olsson O., Koncz C., Szalay A. A. The use of the luxA gene of the bacterial luciferase operon as a reporter gene. Mol Gen Genet. 1988 Dec;215(1):1–9. doi: 10.1007/BF00331295. [DOI] [PubMed] [Google Scholar]
  11. Pruitt R. E., Meyerowitz E. M. Characterization of the genome of Arabidopsis thaliana. J Mol Biol. 1986 Jan 20;187(2):169–183. doi: 10.1016/0022-2836(86)90226-3. [DOI] [PubMed] [Google Scholar]
  12. Reiss B., Sprengel R., Will H., Schaller H. A new sensitive method for qualitative and quantitative assay of neomycin phosphotransferase in crude cell extracts. Gene. 1984 Oct;30(1-3):211–217. doi: 10.1016/0378-1119(84)90122-7. [DOI] [PubMed] [Google Scholar]
  13. St Schell J. Transgenic plants as tools to study the molecular organization of plant genes. Science. 1987 Sep 4;237(4819):1176–1183. doi: 10.1126/science.237.4819.1176. [DOI] [PubMed] [Google Scholar]
  14. Wessel D., Flügge U. I. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem. 1984 Apr;138(1):141–143. doi: 10.1016/0003-2697(84)90782-6. [DOI] [PubMed] [Google Scholar]
  15. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  16. Zambryski P. Basic processes underlying Agrobacterium-mediated DNA transfer to plant cells. Annu Rev Genet. 1988;22:1–30. doi: 10.1146/annurev.ge.22.120188.000245. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES