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Molecular Biology Reports

, Volume 37, Issue 5, pp 2157–2162 | Cite as

The reversed terminator of octopine synthase gene on the Agrobacterium Ti plasmid has a weak promoter activity in prokaryotes

  • Jun-Li Shao
  • Yue-Sheng Long
  • Gu Chen
  • Jun Xie
  • Zeng-Fu XuEmail author
Article
  • 707 Downloads

Abstract

Agrobacterium tumefaciens transfers DNA from its Ti plasmid to plant host cells. The genes located within the transferred DNA of Ti plasmid including the octopine synthase gene (OCS) are expressed in plant host cells. The 3′-flanking region of OCS gene, known as OCS terminator, is widely used as a transcriptional terminator of the transgenes in plant expression vectors. In this study, we found the reversed OCS terminator (3′-OCS-r) could drive expression of hygromycin phosphotransferase II gene (hpt II) and beta-glucuronidase gene in Escherichia coli, and expression of hpt II in A. tumefaciens. Furthermore, reverse transcription-polymerase chain reaction analysis revealed that an open reading frame (ORF12) that is located downstream to the 3′-OCS-r was transcribed in A. tumefaciens, which overlaps in reverse with the coding region of the OCS gene in octopine Ti plasmid.

Keywords

Agrobacterium tumefaciens Antisense overlapping transcripts OCS terminator Promoter activity Hygromycin phosphotransferase II gene (hpt IIBeta-glucuronidase (GUS

Notes

Acknowledgments

We would like to thank an anonymous reviewer for her/his critical and constructive comments that led to the improvement of this article. We thank Dr. Stephen C. Winans (Cornell University, USA) for the A. tumefaciens strains of A348 and R10, and Dr. Peter M. Waterhouse (CSIRO Plant Industry, Australia) for the pHANNIBAL and pART27 plasmids. This work was supported by the National Natural Science Foundation of China (grant nos. 30570943 and 30771111), and the Knowledge Innovation Program of the Chinese Academy of Sciences (grant no. KSCX2-YW-Z-0723) to Z.-F. Xu.

References

  1. 1.
    Zaenen I, Van Larebeke N, Van Montagu M et al (1974) Supercoiled circular DNA in crown-gall inducing Agrobacterium strains. J Mol Biol 86:109–127CrossRefPubMedGoogle Scholar
  2. 2.
    Chilton MD, Drummond MH, Merio DJ et al (1977) Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumorigenesis. Cell 11:263–271. doi: 10.1016/0092-8674(77)90043-5 CrossRefPubMedGoogle Scholar
  3. 3.
    Lemmers M, De Beuckeleer M, Holsters M et al (1980) Internal organization, boundaries and integration of Ti-plasmid DNA in nopaline grown gall tumours. J Mol Biol 144:353–376CrossRefPubMedGoogle Scholar
  4. 4.
    Zambryski P, Holsters M, Kruger K et al (1980) Tumor DNA structure in plant cells transformed by A. tumefaciens. Science 209:1385–1391. doi: 10.1126/science.6251546 CrossRefPubMedGoogle Scholar
  5. 5.
    Klee H, Montoya A, Horodyski F et al (1984) Nucleotide sequence of the tms genes of the pTiA6NC octopine Ti plasmid: two gene products involved in plant tumorigenesis. Proc Natl Acad Sci USA 81:1728–1732CrossRefPubMedGoogle Scholar
  6. 6.
    Korber H, Strizhov N, Staiger D et al (1991) T-DNA gene 5 of Agrobacterium modulates auxin response by autoregulated synthesis of a growth hormone antagonist in plants. EMBO J 10:3983–3991PubMedGoogle Scholar
  7. 7.
    Lichtenstein C, Klee H, Montoya A et al (1984) Nucleotide sequence and transcript mapping of the tmr gene of the pTiA6NC octopine Ti-plasmid: a bacterial gene involved in plant tumorigenesis. J Mol Appl Genet 2:354–362PubMedGoogle Scholar
  8. 8.
    Tinland B, Fournier P, Heckel T et al (1992) Expression of a chimaeric heat-shock-inducible Agrobacterium 6b oncogene in Nicotiana rustica. Plant Mol Biol 18:921–930. doi: 10.1007/BF00019206 CrossRefPubMedGoogle Scholar
  9. 9.
    Guyon P, Chilton MD, Petit A et al (1980) Agropine in “null-type” crown gall tumors: evidence for generality of the opine concept. Proc Natl Acad Sci USA 77:2693–2697CrossRefPubMedGoogle Scholar
  10. 10.
    Zhu J, Oger PM, Schrammeijer B et al (2000) The bases of crown gall tumorigenesis. J Bacteriol 182:3885–3895CrossRefPubMedGoogle Scholar
  11. 11.
    De Greve H, Dhaese P, Seurinck J et al (1982) Nucleotide sequence and transcript map of the Agrobacterium tumefaciens Ti plasmid-encoded octopine synthase gene. J Mol Appl Genet 1:499–511PubMedGoogle Scholar
  12. 12.
    Gleave AP (1992) A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome. Plant Mol Biol 20:1203–1207. doi: 10.1007/BF00028910 CrossRefPubMedGoogle Scholar
  13. 13.
    Jones JD, Shlumukov L, Carland F et al (1992) Effective vectors for transformation, expression of heterologous genes, and assaying transposon excision in transgenic plants. Transgenic Res 1:285–297. doi: 10.1007/BF02525170 CrossRefPubMedGoogle Scholar
  14. 14.
    Wesley SV, Helliwell CA, Smith NA et al (2001) Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J 27:581–590. doi: 10.1046/j.1365-313X.2001.01105.x CrossRefPubMedGoogle Scholar
  15. 15.
    Hood EE, Gelvin SB, Melchers LS et al (1993) New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res 2:208–218. doi: 10.1007/BF01977351 CrossRefGoogle Scholar
  16. 16.
    Sciaky D, Montoya AL, Chilton MD (1978) Fingerprints of Agrobacterium Ti plasmids. Plasmid 1:238–253CrossRefPubMedGoogle Scholar
  17. 17.
    Lyi SM, Jafri S, Winans SC (1999) Mannopinic acid and agropinic acid catabolism region of the octopine-type Ti plasmid pTi15955. Mol Microbiol 31:339–347. doi: 10.1046/j.1365-2958.1999.01178.x CrossRefPubMedGoogle Scholar
  18. 18.
    Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907PubMedGoogle Scholar
  19. 19.
    Barker RF, Idler KB, Thompson DV et al (1983) Nucleotide sequence of the T-DNA region from the Agrobacterium tumefaciens octopine Ti plasmid pTi15955. Plant Mol Biol 2:335–350. doi: 10.1007/BF01578595 CrossRefGoogle Scholar
  20. 20.
    Chung SM, Frankman EL, Tzfira T (2005) A versatile vector system for multiple gene expression in plants. Trends Plant Sci 10:357–361. doi: 10.1016/j.tplants.2005.06.001 CrossRefPubMedGoogle Scholar
  21. 21.
    Boldogkoi Z (2000) Coding in the noncoding DNA strand: a novel mechanism of gene evolution? Journal of Molecular Evolution 51:600–606PubMedGoogle Scholar
  22. 22.
    Williams T, Fried M (1986) A mouse locus at which transcription from both DNA strands produces mRNAs complementary at their 3′ ends. Nature 322:275–279CrossRefPubMedGoogle Scholar
  23. 23.
    Merino E, Balbas P, Puente JL et al (1994) Antisense overlapping open reading frames in genes from bacteria to humans. Nucleic Acids Res 22:1903–1908CrossRefPubMedGoogle Scholar
  24. 24.
    Borsani O, Zhu J, Verslues PE et al (2005) Endogenous siRNAs derived from a pair of natural cis-antisense transcripts regulate salt tolerance in arabidopsis. Cell 123:1279–1291CrossRefPubMedGoogle Scholar
  25. 25.
    Cock JM, Swarup R, Dumas C (1997) Natural antisense transcripts of the S locus receptor kinase gene and related sequences in Brassica oleracea. Mol Genet Genomics 255:514–524CrossRefGoogle Scholar
  26. 26.
    Gelvin SB, Gordon MP, Nester EW et al (1981) Transcription of the agrobacterium Ti plasmid in the bacterium and in crown gall tumors. Plasmid 6:17–29CrossRefPubMedGoogle Scholar
  27. 27.
    Murai N, Kemp JD (1982) Octopine synthase mRNA isolated from sunflower crown gall callus is homologous to the Ti plasmid of Agrobacterium tumefaciens. Proc Natl Acad Sci USA 79:86–90CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Jun-Li Shao
    • 1
  • Yue-Sheng Long
    • 1
    • 3
  • Gu Chen
    • 4
  • Jun Xie
    • 1
  • Zeng-Fu Xu
    • 1
    • 2
    Email author
  1. 1.Key Laboratory of Gene Engineering of the Ministry of Education and State Key Laboratory of Biocontrol, College of Life SciencesSun Yat-sen UniversityGuangzhouChina
  2. 2.Laboratory of Molecular Breeding of Energy Plants, Xishuangbanna Tropical Botanical GardenChinese Academy of SciencesKunmingChina
  3. 3.Institute of Neuroscience and the Second Affiliated Hospital of Guangzhou Medical CollegeGuangzhouChina
  4. 4.College of Light Industry and Food SciencesSouth China University of TechnologyGuangzhouChina

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