생명과학회지 (Journal of Life Science)

  • 제20권9호
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  • Pages.1314-1323
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  • 2010
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  • 1225-9918(pISSN)
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  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
권호 표지
DOI QR코드

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식물에서 표적 유전자의 동시 과발현, 조직/발달 특이적 발현 및 스트레스 유도성 발현을 위한 binary 벡터의 제작과 분석

Construction and Analysis of Binary Vectors for Co-Overexpression, Tissue- or Development-Specific Expression and Stress-Inducible Expression in Plant

  • 이영미 (부산대학교 분자생물학과) ;
  • 박희연 (부산대학교 분자생물학과) ;
  • 우동혁 (부산대학교 분자생물학과) ;
  • 석혜연 (부산대학교 분자생물학과) ;
  • 이선영 (울산대학교 의과대학 생의과학연구소) ;
  • 문용환 (부산대학교 분자생물학과)
  • Lee, Young-Mi (Department of Molecular Biology, Pusan National University) ;
  • Park, Hee-Yeon (Department of Molecular Biology, Pusan National University) ;
  • Woo, Dong-Hyuk (Department of Molecular Biology, Pusan National University) ;
  • Seok, Hye-Yeon (Department of Molecular Biology, Pusan National University) ;
  • Lee, Sun-Young (Ulsan University Hospital Biomedical Research Center, University of Ulsan) ;
  • Moon, Yong-Hwan (Department of Molecular Biology, Pusan National University)
  • 투고 : 2010.07.26
  • 심사 : 2010.09.07
  • 발행 : 2010.09.30
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초록

유전자를 이소성으로 발현하고 억제하는 것은 유전자의 기능 연구에 있어서 매우 유용하다. 본 연구에서는 표적 유전자의 동시 과발현, 조직/발달 단계 특이적 발현 및 스트레스 유도성 발현을 위해 pPZP를 골격으로 다양한 binary 벡터를 제작하고 그 유용성을 검증하였다. 변형된 CaMV 35S 프로모터를 이용하여, 다른 두 개의 유전자를 동시 과발현시키는 binary 벡터를 제작하였고, 이 벡터가 동시에 그리고 같은 장소에서 다른 두 개의 표적 유전자를 과발현 하는데 효과적임을 확인하였다. At2S3, KNAT1 및 LFY 프로모터를 포함하는 조직 또는 발달 단계 특이적 발현 binary 벡터들을 제작하고 분석한 결과, 이 벡터들은 각각 배/유식물 시기, 새싹 끝의 분열조직 및 잎 원기 특이적 발현에 유용하였다. RD29A와 AtNCED3 프로모터를 포함하는 스트레스 유도성 발현 binary 벡터들은 고염, ABA, MV 또는 저온과 같은 비생물성 스트레스에 의한 유전자의 이소성 발현에 유용하였다. 본 연구에서 제작된 binary 벡터들은 표적 유전자의 이소성 발현을 통해 유전자의 생물학적 기능연구, 분자생물학적작용 기작 연구에 유용하게 사용될 것으로 사료된다.

In this study, we constructed various kinds of binary vectors with the pPZP backbone for co-overexpression, tissue- or development-specific expression and stress-inducible expression, and validated them for ectopic expression of target genes. Using a modified CaMV 35S promoter, a binary vector was generated for co-overexpression of two different genes and was confirmed to be efficient for overexpressing two different target genes at the same time and place. Binary vectors containing At2S3, KNAT1 or LFY promoters were constructed for tissue-specific or development-specific gene expression, and the binary vectors were suited for embryo/young seedling stage-, shoot apical meristem- or leaf primordia-specific expressions. Furthermore, the binary vectors containing RD29A or AtNCED3 promoters were validated as suitable vectors for gene expression induced by abiotic stresses such as high salt, ABA, MV and low temperature. Taken together, the binary vectors constructed in this study would be very useful for analyzing the biological functions of target genes and molecular mechanisms through ectopic expression.

키워드

참고문헌

  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. Bevan, M. 1984. Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res. 12, 8711-8721. https://doi.org/10.1093/nar/12.22.8711
  3. Blazquez, M. A., C. Ferrandiz, F. Madueno, and F. Parcy. 2006. How floral meristems are built. Plant Mol. Biol. 60, 855-870. https://doi.org/10.1007/s11103-006-0013-z
  4. Christensen, A. H., R. A. Sharrock, 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
  5. 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
  6. Deblaere, R., A. Reynaerts, H. Hofte, J. P. Hernalsteens, J. Leemans, and M. Van Montagu. 1987. Vectors for cloning in plant cells. Meth. Enzymol. 153, 277-292. https://doi.org/10.1016/0076-6879(87)53059-2
  7. Gallagher, S. R. 1992. GUS protocols: using the GUS gene as a reporter of gene expression. San Diego CA, USA: Academic Press Inc.
  8. Gandhi, R., S. C. Maheshwari, and P. Khurana. 1999. Transient gene expression and influence on foreign gene expression in Arabidopsis thaliana. In Vitro Cell Dev. Biol. Plant 35, 232-237. https://doi.org/10.1007/s11627-999-0084-z
  9. Garbarino, J. E., T. Oosumi, and W. R. Belknap. 1995. Isolation of a polyubiquitin promoter and its expression in transgenic potato plants. Plant Physiol. 109, 1371-1378. https://doi.org/10.1104/pp.109.4.1371
  10. Guerche, P., C. Tire, F. G. De Sa, A. De Clercq, M. Van Montagu, and E. Krebbers. 1990. Differential expression of the Arabidopsis 2S Albumin genes and the effect of increasing gene family size. Plant Cell 2, 469-478. https://doi.org/10.1105/tpc.2.5.469
  11. 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
  12. Hauke, H., M. C. Guitton, and R. Reski. 2002. Plant functional genomics. Naturwissenschaften 89, 235-249. https://doi.org/10.1007/s00114-002-0321-3
  13. 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
  14. Iuchi, S., M. Kobayashi, T. Taji, M. Naramoto, M. Seki, T. Kato, S. Tabata, Y. Kakubari, K. Yamaguchi-Shinozaki, and K. Shinozaki. 2001. Regulation of drought tolerance by gene manipulation of 9-cis-epoxycarotenoid dioxygenase, a key enzyme in abscisic acid biosynthesis in Arabidopsis. Plant J. 27, 325-333. https://doi.org/10.1046/j.1365-313x.2001.01096.x
  15. Jackson, D., B. Veit, and S. Hake. 1994. Expression of maize KNOTTED-1 related homeobox genes in the shoot apical meristem predicts patterns of morphogenesis in the vegetative shoot. Development 120, 405-413.
  16. Kamo, K. K. 2003. Long-term expression of the uidA gene in Gladiolus plants under control of either the ubiquitin, rolD, mannopine synthase, or cauliflower mosaic virus promoters following three seasons of dormancy. Plant Cell Rep. 21, 797-803.
  17. Lee, L. Y. and S. B. Gelvin. 2008. T-DNA binary vectors and systems. Plant Physiol. 146, 325-332. https://doi.org/10.1104/pp.107.113001
  18. Lee, Y. M., H. Y. Seok, H. Y. Park, J. I. Park, J. S. Han, T. S. Bang, and Y. H. Moon. 2009. Construction and verification of useful vectors for ectopic expression and suppression of plant genes. J. Life Sci. 19, 809-817. https://doi.org/10.5352/JLS.2009.19.6.809
  19. Lincoln, C., J. Long, J. Vamaguchi, K. Serikawa, and S. Hake. 1994. A knottedl-like homeobox gene in Arabidopsis is expressed in the vegetative meristem and dramatically alters leaf morphology when overexpressed in transgenic plants. Plant Cell 6, 1859-1876. https://doi.org/10.1105/tpc.6.12.1859
  20. Ma, B. L., K. D. Subedi, and L. M. Reid. 2004. Extent of Cross-Fertilization in Maize by Pollen from Neighboring Transgenic Hybrids. Crop Sci. 44, 1273-1282. https://doi.org/10.2135/cropsci2004.1273
  21. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth bioassays with tobacco tissue cultures. Plant Physiol. 15, 473-497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
  22. Nakashima, K., Y. Fujita, K. Katsura, K. Maruyama, Y. Narusaka, M. Seki, K. Shinozaki, and K. Yamaguchi- Shinozaki. 2006. Transcriptional regulation of ABI3- and ABA-responsive genes including RD29B and RD29A in seeds, germinating embryos, and seedlings of Arabidopsis. Plant Mol. Biol. 60, 51-68. https://doi.org/10.1007/s11103-005-2418-5
  23. Norris, S. R., S. E. Meyer, and J. Callis. 1993. The intron of Arabidopsis thaliana polyubiquitin genes is conserved in location and is a quantitative determinant of chimeric gene expression. Plant Mol. Biol. 21, 895-906. https://doi.org/10.1007/BF00027120
  24. Okamuro, J. K. and R. B. Goldberg. 1989. Regulation of plant gene expression: General principles. In PK stumpf. The Biochemistry of Plants: Academic Press Inc.
  25. Parcy, F., O. Nilsson, M. A. Busch, I. Lee, and D. Weigel. 1998. A genetic framework for floral patterning. Nature 395, 561-566. https://doi.org/10.1038/26903
  26. Plesse, B., M. C. Criqui, A. Durr, Y. Parmentier, J. Fleck, and P. Genchik. 2001. Effects of the polyubiquitin gene Ubi.U4 leader intron and first ubiquitin monomer on reporter gene expression in Nicotiana tabacum. Plant Mol. Biol. 45, 655-667. https://doi.org/10.1023/A:1010671405594
  27. Sakuma, Y., K. Maruyama, Y. Osakabe, F. Qin, M. Seki, K. Shinozaki, and K. Yamaguchi-Shinozaki. 2006. Functional Analysis of an Arabidopsis Transcription Factor, DREB2A, Involved in Drought-Responsive Gene Expression. Plant Cell 18, 1292-1309. https://doi.org/10.1105/tpc.105.035881
  28. Sanchez, R., M. Y. Kim, M. Calonje, Y. H. Moon, and Z. R. Sung. 2009. Temporal and spatial requirement of EMF1 activity for Arabidopsis vegetative and reproductive development. Mol. Plant 2, 643-653. https://doi.org/10.1093/mp/ssp004
  29. Schultz, E. A. and G. W. Haughn. 1991. LEAFY, a homeotic gene that regulates inflorescence development in Arabidopsis. Plant Cell 3, 771-781. https://doi.org/10.1105/tpc.3.8.771
  30. Sheen, J. 2001. Signal Transduction in Maize and Arabidopsis Mesophyll Protoplasts. Plant Physiol. 127, 1466-1475. https://doi.org/10.1104/pp.010820
  31. Shinozaki, K. and K. Yamaguchi-Shinozaki. 2006. Transcriptional regulation of ABI3- and ABA-responsive genes including RD29B and RD29A in seeds, germinating embryos, and seedlings of Arabidopsis. Plant Mol. Biol. 60, 51-68. https://doi.org/10.1007/s11103-005-2418-5
  32. Thomashow, M. F. 1999. Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 571-599. https://doi.org/10.1146/annurev.arplant.50.1.571
  33. Weigel, D. and J. Glazebrook. 2006. Transformation of Agrobacterium using the freeze-thaw method. Cold Spring Harb. Protoc. 10, 1101.
  34. Xiong, L., H. Lee, M. Ishitani, and J. K. Zhu. 2002. Regulation of osmotic stress–`responsive gene expression by the LOS6/ABA1 locus in Arabidopsis. J. Biol. Chem. 277, 8588-8596. https://doi.org/10.1074/jbc.M109275200
  35. Yamaguchi-Shinozaki, K. and K. Shinozaki. 1994. A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salinity stress. Plant Cell 6, 251-264. https://doi.org/10.1105/tpc.6.2.251