Detection of Transgenic Rice Containing CrylAc Gene Derived from Bacillus thuringiensis by PCR

  • Kim, Jae-Hwan (Department of Food Science and Biotechnology, Institute of Life Sciences and Resources, Kyung Hee University) ;
  • Jee, Sang-Mi (Department of Food Science and Biotechnology, Institute of Life Sciences and Resources, Kyung Hee University) ;
  • Park, Cheon-Seok (Department of Food Science and Biotechnology, Institute of Life Sciences and Resources, Kyung Hee University) ;
  • Kim, Hae-Yeong (Department of Food Science and Biotechnology, Institute of Life Sciences and Resources, Kyung Hee University)
  • Published : 2006.08.30

Abstract

Polymerase chain reaction (PCR) method was developed for the specific detection of insect-resistant rice containing cry1Ac gene derived from Bacillus thuringiensis (Bt). Primers were designed from the 35S promoter, NOS terminator, cry1Ac gene, and sucrose phosphate synthase (SPS) for general screening of Bt rice. By sequencing the PCR products from the two putative kinds of Bt rice, we designed a specific primer from the junction region between the cry1Ac gene and the NOS terminator that had been inserted into Bt rice. The construct-specific primer was employed to amplify a 147 bp product in the two lines of Bt rice. No amplified products were observed from the other Bt crops with various Bt genes introduced. In qualitative PCR analysis, the limit of detection was 0.005 ng from genomic DNA of Bt rice. In addition, PCR analysis was performed on 64 kinds of rice presently available in the Korean market, and no Bt rice was detected. This method presented in this paper can be used as a highly sensitive and specific detection method of Bt rice.

Keywords

References

  1. Oh ST, Kim JK, Yang SY, Song MD. Characterization of Bacillus thuringiensis having insecticidal effects against larvae of musca domestica. J. Microbiol. Biotech. 14: 1057-1062 (2004)
  2. Accinelli C, Koskinen WC, Sadowsky MJ. Influence of Cry1Ac Toxin on Mineralization and Bioavailability of Glyphosate in Soil. J. Agr. Food Chem. 54: 164-169 (2006) https://doi.org/10.1021/jf052252v
  3. Cheng X, Sardana R, Kaplan H, Altosaar I. Agrobacterium-transformed rice plants expressing synthetic cryIA(b) and cryIA(c) genes are highly toxic to striped stem borer and yellow stem borer. P. Natl. Acad. Sci. USA 95: 2767-2772 (1998)
  4. Shu Q, Ye G, Cui H, Cheng X, Xiang Y, Wu D, Gao M, Xia Y, Hu C, Sardana R, Altosaar I. Transgenic rice plants with a synthetic cry1Ab gene from Bacillus thuringiensis were highly resistant to eight lepidopteran rice pest species. Mol. Breeding 6: 433-439 (2000) https://doi.org/10.1023/A:1009658024114
  5. Fujimoto H, Itoh K, Yamamoto M, Kyozuka J, Shimamoto K. Insect resistant rice generated by introduction of a modified ${\delta}$-endotoxin gene of Bacillus thuringiensis. Biotechnology (N Y) 11: 1151-1155 (1993)
  6. Tu J, Zhang G, Datta K, Xu C, He Y, Zhang Q, Khush GS, Datta SK. Field performance of transgenic elite commercial hybrid rice expressing Bacillus thuringiensis ${\delta}$-endotoxin. Nat. Biotechnol. 18: 1101-1104 (2002)
  7. Huang J, Hu R, Rozelle S, Pray C. Insect-resistant GM rice in farmers' fields: Assessing productivity and health effects in China. Science 308: 688-690 (2005) https://doi.org/10.1126/science.1108972
  8. James C. Preview: Global Status of Commercialized Biotech/GM Crops. ISAAA 34: 1-11 (2005)
  9. Datta K, Baisakh N, Maung Thet K, Tu J. Pyramiding transgenes for multiple resistance in rice against bacterial blight, yellow stem borer and sheath blight. Theor. Appl. Genet. 106: 1-8 (2002) https://doi.org/10.1007/s00122-002-1014-1
  10. Ceron J, Covarrubias L, Quintero R, Ortiz A, Ortiz M, Aranda E, Lina L, Bravo A. PCR analysis of the cry1 insecticidal crystal family genes from Bacillus thuringiensis. Appl. Environ. Microbiol. 60: 353-356 (1994)
  11. Heo MS, Kim JH, Shin WS, Park SH, Park HK, Kim MC, Kim HY. Limit of detection for genetically modified soybean in doenjang (Korean fermented soy paste). Food Sci. Biotechnol. 13: 657-661 (2004)
  12. Shin DW, Park SH, Woo GJ, Kim HY, Park CS. Case study for natural gene transfer from genetically modified food to food microorganisms. Food Sci. Biotechnol. 13: 342-346 (2004)
  13. McElroy D, Blowers AD, Jenes B, Wu R. Construction of expression vectors based on the rice actin 1 (Actl) 5' region for use in monocot transformation. Mol. Gen. Genet. 231: 150-160 (1991) https://doi.org/10.1007/BF00293832
  14. Heo MS, Kim JH, Park SH, Woo GJ, Kim HY. Detection of genetically modified maize by multiplex PCR method. J. Microbiol. Biotechnol. 14: 1150-1156 (2004)
  15. Ding J, Jia J, Yang L, Wen H, Zhang C, Liu W, Zhang D. Validation of a rice specific gene, sucrose phosphate synthase, used as the endogenous reterence gene for qualitative and real-time quantitative PCR detection of transgenes. J. Agr. Food Chem. 52: 3372-3377 (2004) https://doi.org/10.1021/jf049915d
  16. Yang L, Ding J, Zhang C, Jia J, Weng H, Liu W, Zhang D. Estimating the copy number of transgenes in transtormed rice by real-time quantitative PCR. Plant Cell Rep. 23: 759-763 (2005) https://doi.org/10.1007/s00299-004-0881-0