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Chinese Science Bulletin

, Volume 47, Issue 23, pp 1999–2005 | Cite as

Isolation of a strong matrix attachment region (MAR) and identification of its function in vitro and in vivo

  • Kewei Zhang
  • Jianmei Wang
  • Guodong Yang
  • Xingqi Guo
  • Fujiang Wen
  • Decai Cui
  • Chengchao Zheng
Notes
  • 34 Downloads

Abstract

Inclusion of MARs in transgene cassettes enhances their expression and reduces position-effect variations in the transgenic host. Four new MARs (TM2, TM3, AM1 and AM2) were isolated from tobacco and Arabidopsis by PCR method. The nuclei isolated from suspensioncultured cells of rice were used to prepare nuclear matrix. With a characterized MAR (TM1) as a positive control, the Matrix-MAR interactions were tested by an in vitro binding assay to identify the DNA sequences as MARs and their binding strength to nuclear matrix in vitro was compared. The results showed that TM2 and TM3 had stronger binding strength than TM1. To determine the functions of the four new MARs in vivo, binary vectors pBI121 carrying a uidA GUS reporter gene were modified with direct repeat MARs inserted on both sides of the reporter gene cassette and were transferred into tobaccos via Agrobacterium-mediated transformation procedure. Quantitative GUS assays of the transgenic tobaccos showed that when flanking a GUS reporter gene TM1, TM2, TM3 and AM1 increased uidA GUS gene expression level approximately 1.5-fold, 5-fold, 1.35-fold, 1.3-fold respectively and AM2 has no effect on gene expression. TM2 was found to be a strong MAR that could effectively increase gene expression level and could be used as an effective enhancing element to construct high efficient expression vectors. In this note the relations among the sequence features, binding strength in vitro and function in vivo of the five MARs were analyzed, and the potential significance of TM2 in plant genetic engineering was discussed.

Keywords

MAR isolation nuclear matrix in vitro binding assay β-glucuronidase foreign gene expression high efficient expression vector construction 

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References

  1. 1.
    Bereney, R., Jeon, K. W., Nuclear Matrix Structure and Functional Organization, San Diego: Academic Press, 1995, 22.Google Scholar
  2. 2.
    Katsuya, S., Misa, T., Goshima, S. et al., Presence of an SAR-like sequence in junction regions between an introduced transgene and genomic DNA of cultured tobacco cells: its effect on transformation frequency, Plant J., 2001, 26(4): 375.CrossRefGoogle Scholar
  3. 3.
    Phi-van, L., Strätling, W. H., Dissection of the ability of the chicken lysozyme gene 5′ matrix attachment region to stimulate transgene expression and to dampen position effects, Biochemistry, 1996, 35: 10735.PubMedCrossRefGoogle Scholar
  4. 4.
    Li, X. G., Zhu, Z., Xu, J. W. et al., Isolation of pea matrix attachment region and study on its function in transgenic tobaccos, Science in China, Ser. C., 2001, 31(3): 230.Google Scholar
  5. 5.
    Michalowski, S. M., Allen, G. C., Hall, G. E. et al., Characterization of randomly-obtained matrix attachment regions (MARs) from higher plants, Biochemistry, 1999, 38: 12795.PubMedCrossRefGoogle Scholar
  6. 6.
    Spiker, S., Thompson, W. F., Nuclear matrix attachment regions and transgene expression in plants, Plant Physiol., 1996, 110: 15.PubMedGoogle Scholar
  7. 7.
    Sawasaki, T., Takahashi, M., Goshima, N. et al., Structure of transgene loci in transgenic Arabidopsis plants obtained by particle bombardment: Junction regions can bind to nuclear matrices, Gene, 1998, 218: 27.PubMedCrossRefGoogle Scholar
  8. 8.
    Nomura, K., Saito, W., Moriyana, H., Isolation and characterization of matrix associated region DNA fragments in rice (Oryza sativa L.), Plant Cell Physiol., 1997, 38: 1060.PubMedGoogle Scholar
  9. 9.
    Allen, G. C., Spiker, S., Thompson, W. F., Use of matrix attachment regions (MARs) to minimize transgene silencing, Plant Mol. Biol., 2000, 43: 361.PubMedCrossRefGoogle Scholar
  10. 10.
    Allen, G. C., Hau, G. Jr., Michalowski, S. et al., High-level transgene expression in plant cells: Effects of a strong scaffold attachment region from tobacco, The Plant Cell, 1996, 8: 899.PubMedCrossRefGoogle Scholar
  11. 11.
    Ye, H. C., Study on cell suspension culture and plant regeneration in rice, Acta Bot. Sin., 1984, 26: 52.Google Scholar
  12. 12.
    Masuda, K., Takahshi, S., Nomura, K. et al., A simple procedure for isolation of pure nuclei from carrot embryos in synchronized cultures, Plant Cell Rep., 1991, 10: 329.CrossRefGoogle Scholar
  13. 13.
    Hall, J. R., Allen, G. C., Loer, D. S. et al., Nuclear scaffold and scaffold-attachment regions in higher plants, Proc. Natl. Acad. Sci. USA, 1991, 88: 9320.PubMedCrossRefGoogle Scholar
  14. 14.
    Slatter, R. E., Dupree, P., Gray, J. C., A scaffold-associated DNA region is located downstream of the pea plastocyanin gene, Plant Cell, 1991, 3: 1239.PubMedCrossRefGoogle Scholar
  15. 15.
    Jefferson, R. A., Assaying chimeric genes in plants: The GUS gene fusion system, Plant Mol. Biol. Rep., 1987, 5: 387.CrossRefGoogle Scholar
  16. 16.
    Bradford, M. M., A rapid and sensitive method for the quantitatation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 1976, 72: 248.PubMedCrossRefGoogle Scholar
  17. 17.
    Fagard, M., Vaucheret, H. (trans), Gene silencing in plants: how many mechanisms? Annu, Rev. Plant Physiol. Plant Mol. Biol., 2000, 51: 167.CrossRefGoogle Scholar
  18. 18.
    Breyne, P., Montogu, M., Gheysen, G., The role of scaffold attachment region in the structure and functional organizational of plant chromatin, Transgenic Res., 1994, 3: 195.PubMedCrossRefGoogle Scholar
  19. 19.
    Ülker, B., Allen, G. C., Thompson, W. F. et al., A tobacco matrix attachment region reduces the loss of transgene expression in the progeny of transgenic tobacco plants, Plant J., 1999, 18(3): 253.CrossRefGoogle Scholar
  20. 20.
    Vain, P., Wirlanf, B., Kohli, A. et al., Matrix attachment regions increase expression levels and stability in transgenic rices and their progeny, Plant J., 1999, 18: 233.CrossRefGoogle Scholar
  21. 21.
    Phi-Van, L., Stärtling, W. H., Association of DNA with nuclear matrix, Progr. Mol. Subcell Biol., 1990, 11: 1.Google Scholar
  22. 22.
    Liu, J. W., Tabe, L. M., The influences of two plant nuclear matrix attachment regions (MARs) on gene expression in transgenic plants, Plant Cell Physiol., 1998, 39: 115.PubMedGoogle Scholar

Copyright information

© Science in China Press 2002

Authors and Affiliations

  • Kewei Zhang
    • 1
  • Jianmei Wang
    • 1
  • Guodong Yang
    • 1
  • Xingqi Guo
    • 1
  • Fujiang Wen
    • 1
  • Decai Cui
    • 1
  • Chengchao Zheng
    • 1
  1. 1.College of Life SciencesShandong Agricultural UniversityTai’anChina

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