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애기장대에서 activation tagging system을 이용한 새로운 고염 스트레스 반응 유전자의 동정

Identification of Novel Salt Stress-responsive Genes Using the Activation Tagging System in Arabidopsis

  • 석혜연 (부산대학교 생명시스템학과) ;
  • 응웬부린 (부산대학교 생명시스템학과) ;
  • 배형준 (부산대학교 생명시스템학과) ;
  • 하지민 (부산대학교 생명시스템학과) ;
  • 김하연 (부산대학교 생명시스템학과) ;
  • 이선영 (부산대학교 생명시스템학과) ;
  • 문용환 (부산대학교 생명시스템학과)
  • Seok, Hye-Yeon (Department of Integrated Biological Science, Pusan National University) ;
  • Nguyen, Linh Vu (Department of Integrated Biological Science, Pusan National University) ;
  • Bae, Hyoungjoon (Department of Integrated Biological Science, Pusan National University) ;
  • Ha, Jimin (Department of Integrated Biological Science, Pusan National University) ;
  • Kim, Ha Yeon (Department of Integrated Biological Science, Pusan National University) ;
  • Lee, Sun-Young (Department of Integrated Biological Science, Pusan National University) ;
  • Moon, Yong-Hwan (Department of Integrated Biological Science, Pusan National University)
  • 투고 : 2018.07.30
  • 심사 : 2018.08.23
  • 발행 : 2018.09.30

초록

환경 스트레스는 식물의 성장을 저해하며 작물의 생산량을 감소시키는 주요 원인이다. 식물은 다양한 유전자의 발현 변화를 통해 스트레스에 대한 저항성을 나타낸다. 본 연구에서는 activation tagging system을 이용하여 기존에 밝혀지지 않은 새로운 고염 스트레스 반응 유전자들을 분리하였다. 애기장대의 발아 단계에서 고염 스트레스에 저항성을 보이는 9개의 activation tagging 라인을 선별하였다. 그 중 TAIL-PCR 방법을 이용하여 AT7508, AT7512, AT7527, AT7544, AT7548, AT7556의 6개 라인에서 T-DNA가 삽입된 위치를 확인하였으며 각 라인에서 T-DNA가 삽입된 주변 유전자의 발현을 RT-PCR로 분석하였는데 AT7508, AT7512, AT7527, AT7544, AT7556에서 각각 ClpC2/HSP93-III (At3g48870), plant thionin family (At2g20605), anti-muellerian hormone type-2 receptor (At3g50685), vacuolar iron transporter family protein (At4g27870), microtubule-associated protein (At5g16730)이 activation 된 것으로 밝혀졌다. 더불어 AT7548에서는 T-DNA가 삽입된 곳의 양쪽에 위치하는 두 유전자인 Arabinogalactan protein 13 (AGP13) (At4g26320)과 F-box/RNI-like/FBD-like domains-containing protein (At4g26340)이 모두 activation 되었다. Activation 된 7개 유전자는 기존에 고염 스트레스 저항성과 관련된 기능이 알려지지 않은 유전자로 본 연구를 통해 새롭게 고염 스트레스 반응에 대한 기능이 밝혀졌다. 7개의 activation된 유전자 중 ClpC2/HSP93-III, AGP13, F-box/RNI-like/FBD-like domains-containing protein의 3개 유전자는 고염 스트레스에 의해 발현이 증가하였다. 또한 AT7508과 AT7527, AT7544 라인은 발아 단계뿐만 아니라 유식물체 발달 과정에서도 고염 스트레스 저항성을 보여 activation tagging 라인의 선별 결과의 타당성을 뒷받침 하였다. 본 연구의 결과를 통해 activation tagging system이 새로운 스트레스 반응 유전자를 찾아낼 수 있는 유용한 기술임을 확인할 수 있었다.

Abiotic stresses limit the growth and productivity of plants. Cellular adaptation to abiotic stresses requires coordinated regulation in gene expression directed by complex mechanisms. This study used the activation tagging system to identify novel salt stress-responsive genes. The study selected 9 activation tagging lines that showed salt stress-tolerant phenotypes during their germination stages. Thermal asymmetric interlaced-PCR (TAIL-PCR) was used to identify the T-DNA tagging sites on the Arabidopsis genome in selected activation tagging lines, including AT7508, AT7512, AT7527, AT7544, AT7548, and AT7556. RT-PCR analysis showed that ClpC2/HSP93-III (At3g48870), plant thionin family (At2g20605), anti-muellerian hormone type-2 receptor (At3g50685), vacuolar iron transporter family protein (At4g27870), and microtubule-associated protein (At5g16730) were activated in AT7508, AT7512, AT7527, AT7544, and AT7556, respectively. Interestingly, in AT7548, both the genes adjacent to the T-DNA insertion site were activated: Arabinogalactan protein 13 (AGP13) (At4g26320) and F-box/RNI-like/FBD-like domains-containing protein (At4g26340). All of the seven genes were newly identified as salt stress-responsive genes from this study. Among them, the expression of ClpC2/HSP93-III, AGP13, F-box/RNI-like/FBD-like domains-containing protein gene, and microtubule-associated protein gene were increased under salt-stress condition. In addition, AT7508, AT7527, and AT7544 were more tolerant to salt stress than wild type at seedling development stage, functionally validating the screening results of the activation tagging lines. Taken together, our results demonstrate that the activation tagging system is useful for identifying novel stress-responsive genes.

키워드

참고문헌

  1. Adam, Z., Adamska, I., Nakabayashi, K., Ostersetzer, O., Haussuhl, K., Manuell, A., Zheng, B., Vallon, O., Rodermel, S. R., Shinozaki, K. and Clarke, A. K. 2001. Chloroplast and mitochondrial proteases in Arabidopsis. Plant Physiol. 125, 1912-1918. https://doi.org/10.1104/pp.125.4.1912
  2. Adam, Z., Rudella, A. and van Wijk, K. J. 2006. Recent advances in the study of Clp, FtsH and other proteases located in chloroplasts. Curr. Opin. Plant Biol. 9, 234-240. https://doi.org/10.1016/j.pbi.2006.03.010
  3. Allen, M. D., Kropat, J., Tottey, S., Del Campo, J. A. and Merchant, S. S. 2007. Manganese deficiency in Chlamydomonas results in loss of photosystem II and MnSOD function, sensitivity to peroxides, and secondary phosphorus and iron deficiency. Plant Physiol. 143, 263-277.
  4. Ashraf, M. 2009. Biotechnological approach of improving plant salt tolerance using antioxidants as markers. Biotechnol. Adv. 27, 84-93. https://doi.org/10.1016/j.biotechadv.2008.09.003
  5. Ashraf, M. and Harris, P. J. C. 2004. Potential biochemical indicators of salinity tolerance in plants. Plant Sci. 166, 3-16. https://doi.org/10.1016/j.plantsci.2003.10.024
  6. Constan, D., Froehlich, J. E., Rangarajan, S. and Keegstra, K. 2004. A stromal Hsp100 protein is required for normal chloroplast development and function in Arabidopsis. Plant Physiol. 136, 3605-3615. https://doi.org/10.1104/pp.104.052928
  7. Goussias, C., Boussac, A. and Rutherford, A. W. 2002. Photosystem II and photosynthetic oxidation of water: an overview. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 357, 1369-1381. https://doi.org/10.1098/rstb.2002.1134
  8. Hebbern, C. A., Laursen, K. H., Ladegaard, A. H., Schmidt, S. B., Pedas, P., Bruhn, D., Schjoerring J. K., Wulfsohn, D. and Husted, S. 2009. Latent manganese deficiency increases transpiration in barley (Hordeum vulgare). Physiol. Plant. 135, 307-316. https://doi.org/10.1111/j.1399-3054.2008.01188.x
  9. Ito, T. and Meyerowitz, E. M. 2000. Overexpression of a gene encoding a cytochrome P450, CYP78A9 induces large and seedless fruit in Arabidopsis. Plant Cell 12, 1541-1550. https://doi.org/10.1105/tpc.12.9.1541
  10. Jeong, D. H., An, S., Kang, H. G., Moon, S., Han, J. J., Park, S., Lee, H. S., An, K. and An, G. 2002. T-DNA insertional mutagenesis for activation tagging in rice. Plant Physiol. 130, 1636-1644. https://doi.org/10.1104/pp.014357
  11. Kovacheva, S., Bedard, J., Patel, R., Dudley, P., Twell, D., Rios, G., Koncz, C. and Jarvis, P. 2005. In vivo studies on the roles of Tic110, Tic40, and Hsp93 during chloroplast protein import. Plant J. 41, 412-428.
  12. Lee, S. Y., Seok, H. Y., Tarte, V. N., Woo, D. H., Le, D. H., Lee, E. H. and Moon, Y. H. 2014. The Arabidopsis chloroplast protein S-RBP11 is involved in oxidative and salt stress responses. Plant Cell Rep. 33, 837-847. https://doi.org/10.1007/s00299-013-1560-9
  13. Liu, Y. G., Mitsukawa, N., Oosumi, T. and Whittier, R. F. 1995. Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J. 8, 457-463. https://doi.org/10.1046/j.1365-313X.1995.08030457.x
  14. Marsch-Martinez, N., Greco, R., Van Arkel, G., Herrera-Estrella, L. and Pereira, A. 2002. Activation tagging using the En-I maize transposon system in Arabidopsis. Plant Physiol. 129, 1544-1556. https://doi.org/10.1104/pp.003327
  15. Marschner, H. 2011. Marschner's mineral nutrition of higher plants. 3rd ed., Academic Press: Boston, MA, USA.
  16. Maurizi, M. R. and Xia, D. 2004. Protein binding and disruption by Clp/Hsp100 chaperones. Structure 12, 175-183. https://doi.org/10.1016/j.str.2004.01.021
  17. Merlot, S., Mustilli, A. C., Genty, B., North, H., Lefebvre, V., Sotta, B., Vavasseur, A. and Giraudat, J. 2002. Use of infrared thermal imaging to isolate Arabidopsis mutants defective in stomatal regulation. Plant J. 30, 601-609. https://doi.org/10.1046/j.1365-313X.2002.01322.x
  18. Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473-497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
  19. Nakashima, K., Takasaki, H., Mizoi, J., Shinozaki, K. and Yamaguchi-Shinozaki, K. 2012. NAC transcription factors in plant abiotic stress responses. Biochim. Biophys. Acta. 1819, 97-103. https://doi.org/10.1016/j.bbagrm.2011.10.005
  20. Neuwald, A. F., Aravind, L., Spouge, J. L. and Koonin, E. V. 1999. AAA+: A class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes. Genome Res. 9, 27-43.
  21. Nickelsen, J. and Rengstl, B. 2013. Photosystem II assembly: from cyanobacteria to plants. Annu. Rev. Plant Biol. 64, 609-635. https://doi.org/10.1146/annurev-arplant-050312-120124
  22. Ogura, T. and Wilkinson, A. J. 2001. AAA + superfamily ATPases: common structure-diverse function. Genes Cells 6, 575-597. https://doi.org/10.1046/j.1365-2443.2001.00447.x
  23. Park, H. Y., Seok, H. Y., Park, B. K., Kim, S. H., Goh, C. H., Lee, B. H., Lee, C. H. and Moon, Y. H. 2008. Overexpression of Arabidopsis ZEP enhances tolerance to osmotic stress. Biochem. Biophys. Res. Commun. 375, 80-85. https://doi.org/10.1016/j.bbrc.2008.07.128
  24. Park, S. and Rodermel, S. R. 2004. Mutations in ClpC2/ Hsp100 suppress the requirement for FtsH in thylakoid membrane biogenesis. Proc. Natl. Acad. Sci. USA. 101, 12765-12770. https://doi.org/10.1073/pnas.0402764101
  25. Schirmer, E. C., Glover, J. R., Singer, M. A. and Lindquist, S. 1996. HSP100/Clp proteins: a common mechanism explains diverse functions. Trends Biochem. Sci. 21, 289-296. https://doi.org/10.1016/S0968-0004(96)10038-4
  26. Seok, H. Y., Woo, D. H., Nguyen, L. V., Tran, H. T., Tarte, V. N., Mehdi, S. M. M., Lee, S. Y. and Moon, Y. H. 2017. Arabidopsis AtNAP functions as a negative regulator via repression of AREB1 in salt stress response. Planta 245, 329-341. https://doi.org/10.1007/s00425-016-2609-0
  27. Sjogren, L. L., MacDonald, T. M., Sutinen, S. and Clarke, A. K. 2004. Inactivation of the clpC1 gene encoding a chloroplast Hsp100 molecular chaperone causes growth retardation, leaf chlorosis, lower photosynthetic activity, and a specific reduction in photosystem content. Plant Physiol. 136, 4114-4126. https://doi.org/10.1104/pp.104.053835
  28. Sjogren, L. L. E., Tanabe, N., Lymperopoulos, P., Khan, N. Z., Rodermel, S. R., Aronsson, H. and Clarke, A. K. 2014. Quantitative analysis of the chloroplast molecular chaperone ClpC/Hsp93 in Arabidopsis reveals new insights into its localization, interaction with the Clp proteolytic core, and functional importance. J. Biol. Chem. 289, 11318-11330. https://doi.org/10.1074/jbc.M113.534552
  29. Snider, J., Thibault, G. and Houry, W. A. 2008. The AAA + superfamily of functionally diverse proteins. Genome Biol. 9, 216. https://doi.org/10.1186/gb-2008-9-4-216
  30. Socha, A. L. and Guerinot, M. L 2014. Mn-euvering manganese: the role of transporter gene family members in manganese uptake and mobilization in plants. Front. Plant Sci. 5, 106.
  31. Sreenivasulu, N., Sopory, S. K. and Kavi Kishor, P. B. 2007. Deciphering the regulatory mechanisms of abiotic stress tolerance in plants by genomic approaches. Gene 388, 1-13. https://doi.org/10.1016/j.gene.2006.10.009
  32. Tarte, V. N., Seok, H. Y., Woo, D. H., Le, D. H., Tran, H. T., Baik, J. W., Kang, I. S., Lee, S. Y., Chung, T. and Moon, Y. H. 2015. Arabidopsis Qc-SNARE gene AtSFT12 is involved in salt and osmotic stress responses and Na+ accumulation in vacuoles. Plant Cell Rep. 34, 1127-1138. https://doi.org/10.1007/s00299-015-1771-3
  33. Xiong, L. M., Ishitani, M., Lee, H. and Zhu, J. K. 2001. The Arabidopsis LOS5/ABA3 locus encodes a molybdenum cofactor sulfurase and modulates cold stress- and osmotic stress-responsive gene expression. Plant Cell 13, 2063-2083. https://doi.org/10.1105/tpc.13.9.2063
  34. Zheng, B., Halperin, T., Hruskova-Heidingsfeldova, O., Adam, Z. and Clarke, A. K. 2002. Characterization of chloroplast Clp proteins in Arabidopsis: localization, tissue specificity and stress responses. Physiol. Plant. 114, 92-101. https://doi.org/10.1034/j.1399-3054.2002.1140113.x
  35. Zhu, J. K. 2001. Cell signaling under salt, water and cold stresses. Curr. Opin. Plant Biol. 4, 401-406. https://doi.org/10.1016/S1369-5266(00)00192-8