Development of Salt-Tolerant Transgenic Rice Using Soybean PR10 Gene

콩의 Pathogenesis-Related 10 유전자를 이용한 내염성 벼 형질전환 계통 개발

  • 김효진 (전북대학교 작물생명과학과) ;
  • 백소현 (국립식량과학원 기획조정과) ;
  • 신운철 (국립식량과학원 벼맥류부) ;
  • 서춘순 (국립식량과학원 벼맥류부) ;
  • 박명렬 (전북대학교 농업과학기술연구소) ;
  • 고재권 (국립식량과학원 벼맥류부) ;
  • 윤성중 (전북대학교 작물생명과학과)
  • Received : 2010.11.28
  • Published : 2010.12.31

Abstract

This study was conducted to understand the role of soybean pathogenesis-related 10 (GmPR10) gene in salt tolerance and to develop salt-tolerant rice using GmPR10 cDNA. GmPR10 transgene was expressed constitutively in the shoot and root of the $T_1$ transgenic rice plants. Interestingly, however, the levels of the transgene expression were increased temporally up to over four- to five-fold in the shoot and root by 125 mM NaCl treatment, peaking at six hours after the treatment and decreasing thereafter. Electrolyte leakage of leaf cells under 125 mM NaCl treatment was lower in all the transgenic lines than in the control variety, Dongjin-byeo. Ability of seedlings to recover from 125 mM NaCl treatment for two weeks was higher in the transgenic plants than in the control plants. These results demonstrated that GmPR10 had function to increase cell integrity and promote growth under the saline stress imposed by NaCl. The transgenic line GmPR10-3 which showed highest ability to recover from the saline stress could be used as a potential source for salt tolerance in rice breeding programs.

콩의 PR10 유전자(GmPR10)를 벼에 형질전환하여 GmPR10 전이 유전자의 발현 정도와 내염성 관련 형질의 반응 사이의 인과관계를 조사하여 염 스트레스에 대한 GmPR10 생리적 기능을 분석하고 내염성 유전자원을 개발하였다. 1. 전이 유전자는 형질전환 계통에 따라 게놈 내에 1 ~ 6개의 사본이 도입되었고, 선발된 8개의 형질전환 계통 모두에서 전이 유전자가 발현되었으며, 발현 정도는 계통에 따라 변이를 보였다. 2. $T_1$세대 2계통의 형질전환 식물체와 비 형질전환 식물체에 125mM NaCl을 시간별로 처리한 결과, 전이 유전자 GmPR10의 전사체 검출양은 2계통의 형질전환체에서 모두 염처리 6시간까지 증가하였고, 12시간 이후에는 감소하였다. 3. 세포의 전해질 누출율은 형질전환체가 비 형질전환체에 비해 낮았고, 뿌리가 잎보다 낮았다. 또한, 전이 유전자 전사체의 검출량이 높을수록 전해질 누출율은 낮았다. 4. NaCl 용액에서의 생육 정도는 형질전환체가 비 형질전환체보다 현저히 양호하였으며 GmPR10 전이 유전자의 발현이 높을수록 생육 정도가 더 좋았다. 결론적으로 GmPR10 은 내염성을 증진시키는 기능이 있으며, GmPR10 전이유전자의 발현이 높은 계통은 내염성 벼 육성용 소재로 이용할 수 있을 것으로 평가된다.

Keywords

References

  1. An G 1987. Binary Ti vectors for plant transformation and promoter analysis. Methods Enzymol 153:292-305. https://doi.org/10.1016/0076-6879(87)53060-9
  2. An G, Ebert PR, Mitra A, Ha SB 1988. Binary vectors. In: Plant Molecular Biology Manual A3:1-19. Gelvin SB, Schilperoort R, Verma DP (Eds.) Kluwer Academic Pub, The Netherlands.
  3. Arora R, Pitchay DS, Bearce BC 1998. Water-stress induced heat tolerance in geranium leaf tissues: A possible linkage through stress proteins? Physiol Plant 103:24-34. https://doi.org/10.1034/j.1399-3054.1998.1030104.x
  4. Christensen AB, Cho BH, N$\oe$sby M, Gregersen PL, Brandt J, Madriz-Ordenana K, Collinge DB, Thordal-Christensen H 2002. The molecular characterization of two barley proteins establishes the novel PR-17 family of pathogenesisrelated protein. Mole Plant Pathol 3:135-144. https://doi.org/10.1046/j.1364-3703.2002.00105.x
  5. Dubos C, Plomion C 2001. Drought differentially affects expression of a PR-10 protein in needles of the maritime pine (Pinus pinaster Ait.) seeding. J Exp Bot 52:1143-1144. https://doi.org/10.1093/jexbot/52.358.1143
  6. Dubouzet JG, Sakuma Y, Ito Y, Kasuga M, Dubouzet EG, Miura S, Seki M, Shinizaki K, Yamaguchi-Shinozaki K 2003. OsDREB genes in rice, Oryza sativa L., encode transcription activators that function in drought-, high-saltand cold- responsive gene expression. Plant J 33:751-763. https://doi.org/10.1046/j.1365-313X.2003.01661.x
  7. Koistinen KM, Kokko HI, Hassinen VH, Tervahauta AI, Auriola S, Karenlampi SO 2002. Stress-related RNase PR-10c is post-translationally modified by glutathione in brich. Plant Cell Environ 25:705-715.
  8. Honam agriculture Research Institute 2004. research of salt tolerance in rice. p. 21
  9. Lee KS, Choi SY, Choi WY 1999. Salt Tolerance of Rice during Germination and Early Seedling Stages. Korean J Breed 31:301-305.
  10. Liu J-J, Ekramoddoullah AKM 2006. The family 10 of plant pathogenesis-related proteins : Their structure, regulation, and function in response to biotic and abiotic stresses. Physiol Mol Plant Pathol 68:3-13. https://doi.org/10.1016/j.pmpp.2006.06.004
  11. Maghuly F, Khan MA, Fernadez EB, Druart P, Watillon B, Laimer M 2008. Stress regulated expression of the GUS-marker gene (uidA) under the control of plant calmodulin and viral 35S promoters in a model fruit tress rootstock: Prunnus incise$\times$serrula. Journal of Biotechnology 135:105-116. https://doi.org/10.1016/j.jbiotec.2008.02.021
  12. Moons A, Prinsen E, Bauw G, Van Montagu M 1997. Antagonistic effects of abscisic acid and jasmonates on salt stress-inducible transcripts in rice roots. Plant Cell 9:2243-2259. https://doi.org/10.1105/tpc.9.12.2243
  13. 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
  14. Park CJ, Kim KJ, Shin R, Park JM, Shin YC, Paek KH 2004. Pathogenesis-related protein 10 isolated from hot pepper function as a ribonuclease in an antiviral pathway. Plant J 37:186-198. https://doi.org/10.1046/j.1365-313X.2003.01951.x
  15. Pinto MP, Ricardo CP 1995. Lupinusalbus albus L. Pathogenesis- related proteins that show similarity to PR-10 proteins. Plant Physiol 109:1345-1351. https://doi.org/10.1104/pp.109.4.1345
  16. Pnueli L, Hallak-Herr E, Rozenberg M, Cohen M, Goloubinoff P, Kaplan A, Mittler R 2002. Molecular and biochemical mechanisms associated with dormancy and drought tolerance in the desert legume Retamaraetam. Plant J 31:319-330. https://doi.org/10.1046/j.1365-313X.2002.01364.x
  17. Roger SO, Bendich AJ. (1989) Extraction of DNA from plant tissues : in Plant Molecular Biology Manual, Gelvin SB, Schilperoort R A and Verma D PS. (eds.) pp. A6: 1-10.
  18. Samanani N, Liscombe DK, Facchini PJ 2004. Molecular cloning and characterization of norcoclaurine synthase, an enzyme catalyzing the first committed step in benzylisoquinoline alkaloid biosynthesis. Plant J 40:302-13. https://doi.org/10.1111/j.1365-313X.2004.02210.x
  19. Somssich IE, Schmelzer E, Bollmann JJ. Hahlbrock K 1986. Rapid activation by fungal elicitor of genes encoding 'Pathogenesis-related' protein in cultured parsley cells. Proc Natl Acad Sci USA 83:2427-30. https://doi.org/10.1073/pnas.83.8.2427
  20. Somssich IE, Schmelzer E, Kawalleck P, Hahlbrock K 1988. Gene structure and its in situ transcript localization of pathogenesis-related protein 1 in parsley. Mol Gen Genet 213:93-98. https://doi.org/10.1007/BF00333403
  21. Southern EM 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503-517. https://doi.org/10.1016/S0022-2836(75)80083-0
  22. Utriainen M, Kokko H, Auriola S, Sarrazin O, Karenlampi S 1998. PR-10 protein is induced by copper stress in roots and leaves of a Cu/Zn tolerant clone of bich, Betula pendula. Plant Cell Environ 21:821-828. https://doi.org/10.1046/j.1365-3040.1998.00326.x
  23. Van Loon LC, Pierpoint WS, Boller T, Conejero V 1994. Recommendation for naming plant pathogenesis-related proteins. Plant Mol BiolRep 12:245-264. https://doi.org/10.1007/BF02668748
  24. Van Loon LC. Rep M, Pieterse CMJ 2006. Significance of inducible defense-related proteins in infected plants. Ann Rev Phytopathol 44:7.1-7.28.
  25. Walter MH, Liu JW, Wiinn J, Hess D 1996. Bean ribonuclease- like pathogenesis-related protein genes (Ypr10) display complex patterns of developmental, dark-induced and exogenous-stimulus-dependent expression. Eur J Biochem 239:281-293. https://doi.org/10.1111/j.1432-1033.1996.0281u.x
  26. Wang H, Qi M, Cutler AJ 1993. A simple method of preparing plant samples for PCR. Nucleic Acids Res 21: 4153-4154. https://doi.org/10.1093/nar/21.17.4153
  27. Yoshida S, Forno DA, Cock JH, Gomez KA 1976. Laboratory manual for physiological studies of rice. 3rd edition, The international rice research institute, Philippines. pp. 61-66.