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Detection of transgenes in three genetically modified rice lines by fluorescence in situ hybridization

  • Park, Hye-Mi (Plant Biotechnology Institute, Sahmyook University) ;
  • Jeon, Eun-Jin (Plant Biotechnology Institute, Sahmyook University) ;
  • Waminal, Nomar Espinosa (Plant Biotechnology Institute, Sahmyook University) ;
  • Shin, Kong-Sik (Bio-safety Division, National Academy of Agricultural Science, RDA) ;
  • Kweon, Soon-Jong (Bio-safety Division, National Academy of Agricultural Science, RDA) ;
  • Park, Beom-Seok (Bio-safety Division, National Academy of Agricultural Science, RDA) ;
  • Suh, Seok-Cheol (Bio-safety Division, National Academy of Agricultural Science, RDA) ;
  • Kim, Hyun-Hee (Plant Biotechnology Institute, Sahmyook University)
  • Received : 2010.05.29
  • Accepted : 2010.07.21
  • Published : 2010.12.30

Abstract

Fluorescence in situ hybridization (FISH) using T-DNA probes was applied to localize transgenes onto specific chromosomes and confirm the steady integration of transferred genes in three genetically modified (GM) rice lines, LS28 (event LS30-32-20-1), Cry1Ac1 (event C7-1-9-1) and LS28${\times}$Cry1Ac1 (event L/C1-1-3-1), which are a rice leaf blast-resistant single trait GM line, a leaf folder-resistant single trait GM line, and a rice leaf blast-resistant and leaf folder-resistant stacked GM hybrid line, respectively. The FISH signals were clearly detected on the arms of one homologous chromosome pair for LS28, and on the arms of another chromosome pair for Cry1Ac1 when using the transformation vector pSBM AtCK containing the rice leaf blast-resistant gene (LS28) and pMJ-RTB containing the leaf folder-resistant gene (mCry1Ac1) as a probe, respectively. As expected, we detected two pairs of FISH signals, each on the arms of different chromosome pairs in the stacked GM rice line LS28${\times}$Cry1Ac1 when using both pSBM AtCK and pMJ-RTB as probes. These results indicate that the transgenes are located at specific homologous loci and show position stability among generations in both single trait and stacked GM rice lines. The usefulness and the necessity of FISH to detect inserted genes in transformed plants will be discussed for the purpose of future studies to develop breeding programs and conduct risk assessment of GM plants.

Keywords

References

  1. Abranches R, Santos AP, Wegel E, Williams S, Castilho A, Christou P, Shaw P and Stoger E (2000) Widely separated multiple transgene integration sites in wheat chromosomes are brought together at interphase. Plant J. 24:713-723. https://doi.org/10.1046/j.1365-313x.2000.00908.x
  2. Akiyama H, Sakata K, Kondo K, Tanaka A, Liu MS, Oguchi T, Furui S, Kitta K, Hino A and Teshima R (2008) Individual detection of genetically modified maize varieties in non-identity-preserved maize samples. J. Agric. Food Chem. 56: 1977-1983. https://doi.org/10.1021/jf0727239
  3. Akiyama H, Watanabe T, Wakabayashi K, Nakade S, Yasui S, Sakata K, Chiba R, Spiegelhalter F, Hino A and Maitani T (2005) Quantitative detection system for maize sample containing combined- trait genetically modified maize. Anal. Chem.77: 7421-7428. https://doi.org/10.1021/ac051236u
  4. Castro A and Williams JG (1997) Single-molecule detection of specific nucleic acid sequences in unamplified genomic DNA. Anal. Chem. 69: 3915-3920. https://doi.org/10.1021/ac970389h
  5. Choi HW, Lemaux PG and Cho MJ (2002) Use of fluorescence in situ hybridization for gross mapping of transgenes and screening for homozygous plants in transgenic barley (Hordeum vulgare L.) . Theor. Appl. Genet. 106:92-100. https://doi.org/10.1007/s00122-002-0997-y
  6. Dong JJ, Kharb P, Cervera M, Hall TC (2001) The use of FISH in chromosomal localization of transgenes in rice. Methods Cell Sci. 23: 105-113. https://doi.org/10.1023/A:1013174406432
  7. Harwood WA, Bilham LJ, Travella S, Salvo-Garrido H and Snape JW (2005) Fluorescence in situ hybridization to localize transgenes in plant chromosomes. Methods Mol. Biol. 286: 327-340.
  8. Jackson SA, Zhang P, Chen W, Phillips R, Friebe B, Muthukrishnan S and Gill BS (2001) High-resolution structural analysis of biollistic trans-gene integration into the nuclear genome of wheat. Theor. Appl. Genet. 103: 56-62. https://doi.org/10.1007/s001220100608
  9. James C. (2006) Global status of commecialized biotech/GM crops in 2006. ISAAA Briefs No. 35-2006.
  10. Jiang J, Gill BS, Wang GL, Ronald PC, and Ward DC (1995) Metaphase and interphase fluorescence in situ hybridization mapping of the rice genome with bacterial artificial chromosomes. Proc. Natl. Acad. Sci. USA 92: 4487-4491. https://doi.org/10.1073/pnas.92.10.4487
  11. Jiang J, Hulbert SH., Gill B.S. and Ward DC (1996a) Interphase fluorescence in situ hybridization mapping: a physical mapping strategy strategy for plant species with large complex genomes. Mol. Gen. Genet. 252: 497-502. https://doi.org/10.1007/BF02172395
  12. Jiang J, Nasuda S, Dong F, Scherrer CW, Woo S-S, Wing R, Gill BS and Ward DC. (1996b) A conserved repetitive DNA element located in the centromeres of cereal chromosomes. Proc. Natl. Acad. Sci. USA 93: 14210-14213. https://doi.org/10.1073/pnas.93.24.14210
  13. Jin W-W, Li Z-Y, Fang Q, Altosaar I, Liu L-H and Song J-C (2002) Fluorescence in situ hybridization analysis of alien genes in Agrobacterium-mediated Cry1A(b)-transformed rice. Annals of Botany 90:31-36. https://doi.org/10.1093/aob/mcf160
  14. Kharb P, Dong JJ, Islam-Faridi MN, Stelly DM and Hall TC (2001) Fluorescence in situ hybridization of single copy transgenes in rice chromosomes. In Vitro Cell Dev. Biol. Plant 37:1-5
  15. Kurata N and Omura T (1978) Karyotype analysis in rice - 1. A new method for identifying all chromosome pairs. Jpn. J. Genet 53(4):251-252. https://doi.org/10.1266/jjg.53.251
  16. Lee KR, Shin KS, Suh SC, Kim KY, Jeon YH, Park BS, Kim JK, Kweon SJ and Lee YH (2009) Molecular characterization of lepidopteran pest-resistant transgenic rice events expressing synthetic Cry1Ac. Plant Biotechnol. Rep. 3: 317-324 https://doi.org/10.1007/s11816-009-0105-8
  17. Leggett JM, Perret SJ, Harper J and Morris P. (2000) Chromosomal localization of cotransformed trans-genes in the hexaploid cultivated oat Avena sativa L. using fluorescence in situ hybridization. Heredity 84: 46-53. https://doi.org/10.1046/j.1365-2540.2000.00627.x
  18. Pedersen C, Zimny J, Becker D, Jahne-Gartner A and Lorz H (1997) Localization of introduced genes on the chromosomes of transgenic barley, wheat and triticale by fluorescence in situ hybridization. Theor. Appl. Genet. 94: 749-757. https://doi.org/10.1007/s001220050474
  19. Romano A, Raemakers K, Bernardi J, Visser R and Mooibroek H (2003) Trans-gene organisation in potato after particle bombardment- mediated (co)transformation using plasmids and gene cassettes. Transgenic Res. 12:461-473. https://doi.org/10.1023/A:1024267906219
  20. Salvo-Garrido H, Travella S, Bilham LJ, Harwood WA and Snape JW (2004) The distribution of transgene sites in barley determined by physical and genetic mapping. Genetics 167: 1371-1379. https://doi.org/10.1534/genetics.103.023747
  21. Salvo-Garrido H, Travella S, Schwarzacher T, Harwood WA and Snape JW (2001) An efficient method for the physical mapping of trans-genes in barley using in situ hybridization. Genome 44: 104-110. https://doi.org/10.1139/gen-44-1-104
  22. Santos AP, Wegel E, Allen GC, Thompson WF, Stoger E, Shaw P and Abranches R (2006) In situ methods to localize transgenes and transcripts in interphase nuclei: a tool for transgenic plant research. Plant Methods 2:18. https://doi.org/10.1186/1746-4811-2-18
  23. Shin KS, Lee SM, Lim SH, Woo HJ, Cho HS, Lee KR, Lee MC, Kweon SJ and Suh SC (2009a) Molecular biological characteristics and analysis using the specific markers of leaf folder-resistant GM rice. J. Plant Biotechnol. 36:115-123. https://doi.org/10.5010/JPB.2009.36.2.115
  24. Shin KS, Park JH, Lee JH, Lee SM, Woo HJ, Lim SH, Kim HY, Suh SC and Kweon SJ (2009b) Qualitative PCR detection of stack gene GM rice (LS28XCry1Ac) developed in Korea. J. Appl. Biol. Chem. 52:1-7. https://doi.org/10.3839/jabc.2009.001
  25. Svitashev S, Ananiev E, Pawlowski WP and Somers DA (2000) Association of transgene integration sites with chromosome rearrangements in hexaploid oat. Theor. Appl. Genet. 100:872-880. https://doi.org/10.1007/s001220051364
  26. Travella S, Ross SM, Harden J, Everett C, Snape JW and Harwood WA (2005) A comparison of transgenic barley lines produced by particle bombardment and Agrobacterium-mediated techniques. Plant Cell Rep. 23:780-789. https://doi.org/10.1007/s00299-004-0892-x

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