Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Construction and Verification of Useful Vectors for Ectopic Expression and Suppression of Plant Genes.

식물 유전자의 과발현 및 발현 억제를 위한 유용 벡터의 제조 및 확인

  • Lee, Young-Mi (Department of Molecular Biology, Pusan National University) ;
  • Seok, Hye-Yeon (Department of Molecular Biology, Pusan National University) ;
  • Park, Hee-Yeon (Department of Molecular Biology, Pusan National University) ;
  • Park, Ji-Im (Department of Molecular Biology, Pusan National University) ;
  • Han, Ji-Sung (Department of Molecular Biology, Pusan National University) ;
  • Bang, Tae-Sik (Department of Molecular Biology, Pusan National University) ;
  • Moon, Yong-Hwan (Department of Molecular Biology, Pusan National University)
  • 이영미 (부산대학교 분자생물학과) ;
  • 석혜연 (부산대학교 분자생물학과) ;
  • 박희연 (부산대학교 분자생물학과) ;
  • 박지임 (부산대학교 분자생물학과) ;
  • 한지성 (부산대학교 분자생물학과) ;
  • 방태식 (부산대학교 분자생물학과) ;
  • 문용환 (부산대학교 분자생물학과)
  • Published : 2009.06.30
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Abstract

The phenotypes associated with a gene function are often the best clue to its role in the plant. Transgenic plants ectopically expressing or suppressing a gene can provide useful information related to the gene function. In this study, we constructed three vectors - pFGL571, pFGL846 and pFGL847 - for the Agrobacterium-mediated ectopic expression of plant genes using pPZP211 and modified CaMV 35S, UBQ3 or UBQ10 promoters. The three vectors have several merits such as small size, high copy in bacteria, enough restriction enzyme sites in multi cloning sites and nucleotide sequence information. Analysis of transgenic plants containing GUS or sGFP reporter genes under the control of modified CaMV 35S, UBQ3 or UBQI0 promoter revealed that all of the three promoters showed high activities during most developmental stages after germination and in floral organs. Furthermore, we generated a RNAi module vector, pFGL727, to suppress plant gene expressions and confirmed that pFGL727 is useful for the suppression of a gene expression using rice transgenic plants. Taken together, our new vectors would be very useful for the ectopic expression or the suppression of plant genes.

식물에서 유전자의 기능을 연구하는데 있어서 유전자가 과발현 되거나 발현이 억제되는 형질전환체는 해당 유전자의 기능과 관련되어 매우 유용한 정보를 제공한다. 본 연구에서는 modified CaMV 355, UBQ3, UBQ10 프로모터를 pPZP211 벡터에 각각 클로닝 하여 Agrobacterium을 매개로 한 과발현형질전환 식물체 제작에 유용하게 이용할 수 있는 pFGL571, pFGL846, pFGL847을 제조하였다. 이 벡터들은 크기가 작고, 박테리아 내에 high copy로 존재하며, 다중 클로닝 부위에 다양한 제한효소 부위를 가지고 있고, 전체 서열이 알려져 있는 등의 장점을 가지고 있다. GUS 또는 sGFP 리포터 유전자를 포함하는 형질전환 식물체를 제조하여 modified CaMV 35S, UBQ3, UBQ10 프로모터의 활성을 분석한 결과, 세 프로모터 모두 발아 후 대부분의 발달단계와 성숙한 식물체의 꽃 기관에서 높은 활성을 보였다. 한편, 식물에서 유전자 발현 억제에 이용할 수 있는 RNAi 기본 벡터인 pFGL727을 제조하였고, pFGL727을 이용한 벼 RNAi 형질전환체의 분석을 통해 이벡터가 유전자의 발현 억제에 유용하게 이용될 수 있음을 확인하였다. 연구 결과를 종합해 보면, 본 연구에서 제조한 벡터들은 식물에서 유전자 과발현과 발현 억제에 유용하게 이용될수 있을 것으로 기대된다.

Keywords

References

  1. An, G. 1986. Development of plant promoter expression vectors and their use for analysis of differential activity of nopaline synthase promoter in transformed tobacco tissue. Plant Physiol. 81, 86-91 https://doi.org/10.1104/pp.81.1.86
  2. Benfey, P. N., L. Ren, and N. H. Chua. 1989. The CaMV 35S enhancer contains at least two domains which can confer different developmental and tissue-specific expression patterns. EMBO J. 8, 2195-2202
  3. Benfey, P. N., L. Ren, and N. H. Chua. 1990a. Tissue-specific expression from CaMV 35S enhancer subdomains in early stages of plant development. EMBO J. 9, 1677-1684
  4. Benfey, P. N., L. Ren, and N. H. Chua. 1990b. Combinatorial and synergistic properties of CaMV 35S enhancer subdomains. EMBO J. 9, 1685-1696
  5. Bevan, M. 1984. Binary Agrobacterium vectors for plant transformation. Nucl. Acids Res. 12, 8711-8721 https://doi.org/10.1093/nar/12.22.8711
  6. Callis, J., T. Carpenter, C. W. Sun, and R. D. Vierstra. 1995. Structure and Evolution of Genes Encoding Polyubiquitin and Ubiquitin-Like Proteins in Arabidqpsis thaliana Ecotype Columbia. Genetics 159, 921-939
  7. Chalfie, M., Y. Tu, G. Euskirchen, W. W. Ward, and D. C. Prasher. 1994. Green fluorescent protein as a marker for gene expression. Science 263, 802-805 https://doi.org/10.1126/science.8303295
  8. Christensen, A. H., R. A. Sharrok, and P. H. Quail. 1992. Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Plant Mol. Biol. 18, 675-689 https://doi.org/10.1007/BF00020010
  9. Clough, S. J. and A. F. Bent. 1998. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735-743 https://doi.org/10.1046/j.1365-313x.1998.00343.x
  10. Fang, R. X., F. Nagy, S. Sivasubramaniam, and N. H. Chua. 1989. Multiple cis Regulatory Elements for Maximal Expression of the Cauliflower Mosaic Virus 35S Promoter in Transgenic Plants. Plant Cell 1, 141-150 https://doi.org/10.1105/tpc.1.1.141
  11. Gallie, D. R., D. E. Sleat, J. W. Watts, P. C. Turner, and T. M. A. Wilson. 1987. A comparison of eukaryotic viral 5'-reader sequences as enhancers of mRNA expression in vivo. Nucl. Acids Res. 15, 8693-8711 https://doi.org/10.1093/nar/15.21.8693
  12. Gao, P., Z. Xin, and Z. L. Zheng. 2008. The OSU1/QUA2/TSD2-Encoded Putative Methyltransferase Is a Critical Modulator of Carbon and Nitrogen Nutrient Balance Response in Arabidopsis. PLoS ONE 3, e1387 https://doi.org/10.1371/journal.pone.0001387
  13. Hajdukiewicz, P., Z. Svab, and P. Maliga. 1994. The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol. Biol. 25, 989-994 https://doi.org/10.1007/BF00014672
  14. Harholt, J., J. K. Jensen, S. O. Sørensen, C. Orfila, M. Pauly, and H. V. Scheller. 2006. ARABINAN DEFICIENT 1 is a putative arabinosyltransferase involved in biosynthesis of pectic arabinan in arabidopsis. Plant Physiol. 140, 49-58 https://doi.org/10.1104/pp.105.072744
  15. Herrera-Estrella, L., M. D. Block, E. Messens, J. P. Hernalsteens, M. V. Montagu, and J. Schell. 1983. Chimeric genes as dominant selectable markers in plant cells. EMBO J. 2, 987-995
  16. Hiei, Y., T. Komari, and T. Kubo. 1997. Transformation of rice mediated by Agrobacterium tumefaciens. Plant Mol. Biol. 35, 205-218 https://doi.org/10.1023/A:1005847615493
  17. Hofgen, R. and L. Willmitzer. 1988. Storage of competent cells for Agrobacterium transformation. Nucl. Acids Res. 18, 9877
  18. Holtorf, H., M. C. Guitton, and R. Reski. 2002. Plant functional genomics. Naturwissenschaften 89, 235-249 https://doi.org/10.1007/s00114-002-0321-3
  19. Holtorf, S., K. Apel, and H. Bohlmann. 1995. Comparison of different constitutive and inducible promoters for the overexpression of transgenes in Arabidopsis thaliana. Plant Mol. Biol. 29, 637-646 https://doi.org/10.1007/BF00041155
  20. Kusaba, M. 2004. RNA interference in crop plants. Curr. Opin. Biotechnol. 15, 139-143 https://doi.org/10.1016/j.copbio.2004.02.004
  21. Kusaba, M., K. Miyahara, S. Iida, H. Fukuoka, T. Takano, H. Sassa, M. Nishimura, and T. Nishio. 2003. Low glutelin content 1: a dominant mutation that suppresses the glutelin multigene family via RNA silencing in rice. Plant Cell 15, 1455-1467 https://doi.org/10.1105/tpc.011452
  22. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth bioassays with tobacco tissue cultures. Physiol. Plant 15, 473-497 https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
  23. Park, H. Y., I. S. Kang, J. S. Han, C. H. Lee, G. An, and Y. H. Moon. 2009. OsDEG10 encoding a small RNA-binding protein is involved in abiotic stress signaling. Biochem. Biophys. Res. Commun. 380, 597-602 https://doi.org/10.1016/j.bbrc.2009.01.131
  24. Pereira, A. 2000. A transgenic perspective on plant functional genomics. Transgenic Res. 9, 245-260 https://doi.org/10.1023/A:1008967916498
  25. Sanders, P. R., J. A. Winter, A. R. Bamason, S. G. Rogers, and R. T. Fraley. 1987. Comparison of cauliflower mosaic virus 35S and nopaline synthase promoters in transgenic plants. Nucl. Acids Res. 15, 1543-1558 https://doi.org/10.1093/nar/15.4.1543
  26. Shabalina, S. A. and E. V. Koonin. 2008. Origins and evolution of eukaryotic RNA interference. Trends Ecol. Evol. 23, 578-587 https://doi.org/10.1016/j.tree.2008.06.005
  27. Smith, N. A., S. P. Singh, M. B. Wang, P. A. Stoutjesdijk, A. G. Green, and P. M. Waterhouse. 2000. Total silencing by intron-spliced hairpin RNAs. Nature 407, 319-320 https://doi.org/10.1038/35030305
  28. Stoutjesdijk, P. A., S. P. Singh, Q. Liu, C. J. Hurlstone, P. A. Waterhouse, and A. G. Green. 2002. hpRNA-mediated targeting of the Arabidopsis FAD2 gene gives highly efficient and stable silencing. Plant Physiol. 129, 1723-1731 https://doi.org/10.1104/pp.006353
  29. Sun, C. W. and J. Callis. 1997. Independent modulation of Arabidopsis thaliana polyubiquitin mRNAs in different organs and in response to environmental changes. Plant J. 11, 1017-1027 https://doi.org/10.1046/j.1365-313X.1997.11051017.x
  30. Wally, O., J. Jayaraj, and Z. K. Punja. 2008. Comparative expression of beta-glucuronidase with five different promoters in transgenic carrot (Daucus carota L.) root and leaf tissues. Plant Cell Rep. 27, 279-287 https://doi.org/10.1007/s00299-007-0461-1
  31. Wang, J. and J. H. Oard. 2003. Rice ubiquitin promoters: deletion analysis and potential usefulness in plant transformation systems. Plant Cell Rep. 22, 129-134 https://doi.org/10.1007/s00299-003-0657-y
  32. Weltmeier, F., F. Rahmani, A. Ehlert, K. Dietrich, K. Schutze, X. Wang, C. Chaban, J. Hanson, M. Teige, K. Harter, J. Vicente-Carbajosa, S. Smeekens, and W. Droge-Laser. 2009. Expression patterns within the Arabidopsis C/S1 bZIP transcription factor network: availability of heterodimerization partners controls gene expression during stress response and development. Plant Mol. Biol. 69, 107-119 https://doi.org/10.1007/s11103-008-9410-9
  33. Wesley, S. V., C. A. Helliwell, N. A. Smith, M. B. Wang, D. T. Rouse, Q. Liu, P. S. Gooding, S. P. Singh, D. Abbott, and P. A. Stoutjesdijk. 2001. Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J. 27, 581-590 https://doi.org/10.1046/j.1365-313X.2001.01105.x
  34. Zhang, H., C. Ransom, P. Ludwig, and S. van Nocker. 2003. Genetic Analysis of Early Flowering Mutants in Arabidopsis Defines a Class of Pleiotropic Developmental Regulator Required for Expression of the Flowering-Time Switch Flowering Locus C. Genetics 164, 347-358
  35. Zheng, X., W. Deng, K. Luo, H. Duan, Y. Chen, R. McAvoy, S. Song, Y. Pei, and Y. Li. 2007. The cauliflower mosaic virus (CaMV) 35S promoter sequence alters the level and patterns of activity of adjacent tissue- and organ-specific gene promoters. Plant Cell Rep. 26, 1195-1203 https://doi.org/10.1007/s00299-007-0307-x

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