Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Cloning and Expression Analysis of a Grape asr gene, VlASR Containing a Promoter Region.

포도 VIASR 유전자 프로모터의 분리 및 발현 분석

  • Kihl, Joon-Yeong (Department of Molecular Biology and Institute of Nanosensor and Biotechnology, Dankook University) ;
  • Pyee, Jae-Ho (Department of Molecular Biology and Institute of Nanosensor and Biotechnology, Dankook University)
  • 길준영 (단국대학교 분자생물학과 / 나노센서바이오텍연구소) ;
  • 피재호 (단국대학교 분자생물학과 / 나노센서바이오텍연구소)
  • Published : 2007.08.30
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Abstract

VvMSA, a grapevine ASR which is highly inducible by sugar and abscisic acid signals was previously shown to be a transcription factor for a hexose transporter gene VvHT1. We isolated a cDNA clone, VlASR which is regulated temporally during the grape berry development by ACP RT-PCR (annealing control primer reverse transcriptase-polymerase chain reaction) and it proved identical to VvMSA. RT-PCR and real-time PCR analyses revealed that the VlASR gene was expressed in berries at fruit set and that its expression increased as berries aged but decreased at the late ripening stage. In order to understand the regulatory mechanism of the asr gene, a genomic fragment was cloned from grapevine. The genomic DNA was 1375 bp long and a sugar box (sucrose box 3 and sucrose responsive element 1) was identified in the 611 bp upstream region of the open reading frame. Analysis of the VlASR promoter::reporter gene fusion demonstrated that this promoter was expressed in transgenic Arabidopsis even without sucrose treatment. This result suggests that the ASR/VvHT1-mediated sugar/ABA signaling, previously reported in grapevine, may not function in Arabidopsis which has no ASR homologue.

포도 ASR (VvMSA) 단백질은 hexose transporter 유전자 VvHT1의 전사를 조절하는 조절 인자 중의 하나로서 sugar 및 abscisic acid (ABA) 신호에 의해 발현이 유도된다. 본 연구진은 ACP RT-PCR (annealing control primer reverse transcriptase-polymerase chain reaction) 방법을 이용하여 포도 과실발달 과정에서 조절되는 유전자 중 VvMSA와 동일한 cDNA (VlASR)를 클로닝하였다. 이 유전자는 착과 시기에 발현되기 시작하여 과실이 발달하면서 점점 증가하여 착과 후 10 주에 가장 많이 발현되며, 숙기 후반에는 도리어 발현양이 감소하였다. 포도 asr 유전자의 조절기작을 밝히기 위해, 이 유전자의 genomic clone을 분리하였다. 총 1375 bp로 이루어진 이 유전자 절편에는 open reading frame과 100 bp의 intron을 포함하고 있다. 약 600 bp 길이의 프로모터 내에는 sugar 신호전달과 연관이 있는 것으로 알려진 sugar box(sucrose box 3 +sucrose response box 1)가 있다. 프로모터 절편을 reporter 유전자와 연결하여 Arabidopsis에 도입하고 형질전환체를 분석한 결과, reporter 유전자는 sucrose 처리와 상관없이 항상 발현되었다. 이러한 결과는 포도에서 보고된 ASR/VvHT1를 매개로 하는 sugar/ABA 신호전달계가 asr 유전자가 없는 Arabidopsis에서는 작동되지 않음을 시사하고 있다.

Keywords

References

  1. Amitai-Zeigerson, H., P. A. Scolnik and D. Bar-Zvi. 1994. Genomic nucleotide sequence of tomato asr2, a second member of the sress/ripening-induced asr1 gene family. Plant Physiol. 106, 1699-1700. https://doi.org/10.1104/pp.106.4.1699
  2. Cakir, B., A. Agasse, C. Gaillard, A. Saumonneau, S. Delrot and R. Atanassova. 2003. A grape ASR protein involved in sugar and abscisic acid signaling. The Plant Cell 15, 2165-2110. https://doi.org/10.1105/tpc.013854
  3. Canel, C., J. N. Bailey-Serres and M. L. Roose. 1995. Pummelo fruit transcript homologous to ripening-induced genes. Plant Physiol. 108, 1323-1325. https://doi.org/10.1104/pp.108.3.1323
  4. Chang, S., J. D. Puryear, D. M. Dias, E. A. Funkhouser, R. J. Newton and J. Cairney. 1996. Gene expression under water deficit in loblolly pine (Pinus taeda L.): isolation and characterization of eDNA clones. Physiol. Platarum 97, 139-148. https://doi.org/10.1111/j.1399-3054.1996.tb00490.x
  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. Coombe, B. G. 1992. Research on development and ripening of the grape berry. Am. J. Enol. Vitic. 43, 101-110.
  7. Doczi, R., C. Csanaki and Z. Banfalvi. 2002. Expression and promoter activity of the desiccation-specific Solanum tuberosum gene, StDS2. Plant Cell and Environment 25, 1197-1203. https://doi.org/10.1046/j.1365-3040.2002.00904.x
  8. Doyle, J. J. and J. L. Doyle. 1990. Isolation of plant DNA from fresh tissue. Focus 12, 13-15.
  9. Finkelstein, R. R. and S. I. Gibson. 2002. ABA and sugar interactions regulating development: cross-talk or voices in a crowd? Curr. Opin. Plant Biol. 5, 26-32. https://doi.org/10.1016/S1369-5266(01)00225-4
  10. Grierson, C., J. S. Du, S. De Torres Zabala, K. Beggs, C. Smith, M. Holsworth and M. Bevan. 1994. Separate cis sequences and trans factors direct metabolic and developmental regulation of a potato tuber storage protein gene. Plant J. 5, 815-826. https://doi.org/10.1046/j.1365-313X.1994.5060815.x
  11. Hattori, T., S. Nakagawa and K. Nakamura. 1990. High level expression of tuberous root storage protein genes of sweet potato in sterns of plantlets grown in vitro on sucrose medium. Plant Mol. Biol. 14, 595-604. https://doi.org/10.1007/BF00027505
  12. Higo, K., Y. Ugawa, M. Iwamoto and T. Korenaga. 1999. Plant cis-acting regulatory DNA elements (PLACE) databases: 1999. Nucl. Acids Res. 27, 297-300. https://doi.org/10.1093/nar/27.1.297
  13. Hoefgen, R. and L. Willmitzer. 1988. Storage of competent cells for Agrobacteriunl transformation. Nucl. Acids Res. 16, 9877. https://doi.org/10.1093/nar/16.20.9877
  14. Hong, S. H., I. J. Kim, D. C. Yang and W. I. Chung. 2002. Characterization of an abscisic acid responsive gene homologue from Cucumis melo. J. Exp. Bot. 53, 2271-2272. https://doi.org/10.1093/jxb/erf075
  15. Hrazdina, G., C. F. Parsons and L. R. Mattick. 1984. Physiological and biochemical events during development and maturation of grape berries. Am. J. Enol. Vitic. 35, 220-227.
  16. Huang, J. C., S. M. Lin and C. S. Wang. 2000. A pollen-specific and desiccation associated transcript in Lilium longif1orum during development and stress. Plant Cell Physiol. 41, 477-485. https://doi.org/10.1093/pcp/41.4.477
  17. Itai, A., K. Tanabe, F. Tamura and T. Tanaka. 2000. Isolation of cDNA clones corresponding to genes expressed during fruit ripening in Japanese pear (Pyrus pyrifolia Nakai): involvement of the ethylene signal transduction pathway in their expression. J. Exp. Bot. 51, 1163-1166. https://doi.org/10.1093/jexbot/51.347.1163
  18. Iusem, N. D., D. M. Bartholomew, W. D. Hitz and P. A. Scolnik. 1993. Tomato (Lycopersicon esculentum) transcript induced by water deficit and ripening. Plant Physiol. 102, 1353-1354. https://doi.org/10.1104/pp.102.4.1353
  19. Jefferson, R. A. 1987. Assaying chimeric genes in plants: the GUS gene fusion system. Plant Molecular Biology Reporters 5, 387-405. https://doi.org/10.1007/BF02667740
  20. Lucille, A. and G. Don. 2002. Ethylene biosynthesis and action in tomato: a model for climacteric fruit ripening. J. Exp. Bot. 53, 2039-2055. https://doi.org/10.1093/jxb/erf072
  21. Maskin, L., E. Gustavo, G. Gudesblat, J. E. Moreno, F. Carrari, N. Frankel, A. Sambade, M. Rossi and N. D. Iusem. 2001. Differential expression of the members of the asr gene family in tomato (Lycopersicon esculentum). Plant Sci. 161, 739-746. https://doi.org/10.1016/S0168-9452(01)00464-2
  22. Maskin, L., N. Frankel, G. Gudesblat, M. J. Demergasso, L. I. Pietrasanta and N. D. Iusem. 2007. Dimerization and DNA-binding of ASR1, a small hydrophilic protein abundant in plant tissues suffering from water loss. Biochemical and Biophysical Research Communications 352, 831-835. https://doi.org/10.1016/j.bbrc.2006.11.115
  23. Maskin, L., S. Maldonado and N. D. Iusem. 2007. Tomato leaf spatial expression of stress-induced ASR genes. Mol. Biol. Rep. Epub ahead of print. 2007 Jun 30.
  24. Mbeguie-A-Mbeguie, D., R. M. Gomez and B. Fils-Lycaon. 1997. Molecular cloning and nucleotide sequence of an abscisic acid-, stress-, ripening-induced (ASR)-like protein from apricot fruit. Plant Physiol. 115, 1287-1289. https://doi.org/10.1104/pp.115.3.1287
  25. Meyer, P. 1995. Understanding and controlling transgene expression. Trends in Biotechnol. 13, 332-337. https://doi.org/10.1016/S0167-7799(00)88977-5
  26. Padarnanabhan, V., D. M. Dias and R. J. Netwon. 1997. Expression analysis of a gene family in loblolly pine (Pinus taeda L.) induced by water deficit sress. Plant Mol. Biol. 35, 801-807. https://doi.org/10.1023/A:1005897921567
  27. Pla, M., J. Vilardell, M. J. Guiltinan, W. R. Marcotte, M. F. Niogret, R. S. Quatrano and M. Pages. 1993. The cis-regulatory element CCACGTGG is involved in ABA and water-stress responses of the maize gene rab28. Plant Mol. Biol. 21, 259-266. https://doi.org/10.1007/BF00019942
  28. Rombauts, S., P. Dehais, M. Van Montagu and P. Rouze. 1999. PlantCARE, a plant cis-acting regulatory element databases. Nucl. Acids Res. 27, 295-296. https://doi.org/10.1093/nar/27.1.295
  29. Rossi, M. and N. D. Tusem. 1994. Tomato (Lycopersicon esculentum) genomic clone homologous to a gene encoding an abscisic acid-induced protein. Plant Physiol. 104, 1073-1074. https://doi.org/10.1104/pp.104.3.1073
  30. Sambrook, J. and D. W. Russel. 2001. Molecular Cloning: A laboratory Manual. pp. 7.35-7.40, 3rd eds., Cold Spring Harbor Laboratory Press, New York.
  31. Silhavy, D., G. Hutvagner, E. Barta and Z. Banfalvi. 1995. Isolation and characterization of a water-stress-inducible cDNA clone from Solanum chacoense. Plant Mol. Biol. 27, 587-595. https://doi.org/10.1007/BF00019324
  32. Stephanie, K. C. and G. D. May. 1997. Differential gene expression in ripening banana fruit. Plant Physiol. 115, 463-469. https://doi.org/10.1104/pp.115.2.463
  33. Vaidyanathan, R., S. Kuruvila and G. Thomas. 1999. Characterization and expression pattern of an abscisic acid and osmotic stress responsive gene from lice. Plant Sci. 140, 21-30. https://doi.org/10.1016/S0168-9452(98)00194-0
  34. Vrebalov, J., D. Ruezinsky, V. Padmanabhan, R. White, D. Medrano, R. Drake, W. Schuch and J. Giovannoni. 2002. A MADS-box gene necessary for fruit ripening at the tomato ripening-inhibitor (rin) locus. Science 296, 343-346. https://doi.org/10.1126/science.1068181
  35. Wang, C. S., Y. E. Liau, J. C. Huang, T. D. Wu, C. C. Su and C. H. Lin. 1998. Characterization of a desiccation-related protein in lily pollen during development and stress. Plant Cell Physiol. 39, 307-1314. https://doi.org/10.1093/oxfordjournals.pcp.a029371
  36. Welbaum, G. E., T. Tissaoui and K. J. Bradford. 1990. Water relations of seed development and germination in muskmelon (Cucumis meio L.) : III. Sensitivity of germination to water potential and abscisic acid during development. Plant Physiol. 92, 1029-1037. https://doi.org/10.1104/pp.92.4.1029
  37. Yazaki, K. 2005. Transporters of secondary metabolites. Curr. Opin. Plant Biol. 8, 301-307. https://doi.org/10.1016/j.pbi.2005.03.011