Characterization, detection and identification of transgenic chili pepper harboring coat protein gene that enhances resistance to cucumber mosaic virus
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Previously, two events (H15 and B20) of transgenic pepper (Capsicum annuum L.) that enhanced resistance to Cucumber mosaic virus (CMV) by the introduction of CMV coat protein (CP) gene were constructed. Presently, a single copy number of the CP gene was revealed in H15 and B20 by Southern blot. To predict possible unintended effects due to transgene insertion in an endogenous gene, we carried out sequencing of the 5’-flanking region of the CP gene and a Blast- based search. The results revealed that insertion of the transgene into genes encoding putative proteins may occur in the H15 and B20 transgenic event. Mutiplex polymerase chain reaction (PCR) for simultaneous detection and identification of transgenic pepper was conducted with a set of nine primers. Both transgenic event were differentiated from non-transgenic event by the presence of 267 bp and 430 bp PCR products indicative of CP gene specific primer pairs and primer pairs targeting the CP gene and 35S promoter. H15 and B20 uniquely possessed a 390 bp and 596 bp PCR product, respectively. The presence of a 1115 bp product corresponding to intrinsic pepper actin gene confirmed the use of pepper DNA as the PCR template. The primer set and PCR conditions used presently may allow the accurate and simple identification of CMV resistant transgenic pepper.
Abstract
Previously, two events (H15 and B20) of transgenic pepper (Capsicum annuum L.) that enhanced resistance to Cucumber mosaic virus (CMV) by the introduction of CMV coat protein (CP) gene were constructed. Presently, a single copy number of the CP gene was revealed in H15 and B20 by Southern blot. To predict possible unintended effects due to transgene insertion in an endogenous gene, we carried out sequencing of the 5’-flanking region of the CP gene and a Blast- based search. The results revealed that insertion of the transgene into genes encoding putative proteins may occur in the H15 and B20 transgenic event. Mutiplex polymerase chain reaction (PCR) for simultaneous detection and identification of transgenic pepper was conducted with a set of nine primers. Both transgenic event were differentiated from non-transgenic event by the presence of 267 bp and 430 bp PCR products indicative of CP gene specific primer pairs and primer pairs targeting the CP gene and 35S promoter. H15 and B20 uniquely possessed a 390 bp and 596 bp PCR product, respectively. The presence of a 1115 bp product corresponding to intrinsic pepper actin gene confirmed the use of pepper DNA as the PCR template. The primer set and PCR conditions used presently may allow the accurate and simple identification of CMV resistant transgenic pepper.
참고문헌 (14)
- Ahmed FE, "Detection of genetically modified organisms in foods", Trends in Biotech, 20, pp.215~223 (2002)
- Ezequiel M, "Detection of BT transgenic maize in foodstuffs", Food Res Inter, 39, pp.250~255 (2006)
- Germini A, "Development of a seven-target multiplex PCR for the simultaneous detection of transgenic soybean and maize in feeds and foods", J Agric Food Chem, 52, pp.3275~3280 (2004)
- Hamill JD, "The use of polymerase chain reaction in the plant transformation studies", Plant Cell Rep, 10, pp.221~224 (1991)
- Henegariu O, "Multiplex PCR: critical parameters and step-by-step protocol", BioTech, 23, pp.504~511 (1997)
- Global status of commercialized biotech, "GM crops", James C, 2005, ISAAA Briefs NO 34
- James D, "Reliable detection and identification of genetically modified maize, soybean, and canola by multiplex PCR analysis", J Agric Food Chem, 51, pp.5829~5834 (2003)
- Kim SH, "Rapid and reliable method of extracting DNA and RNA from sweet potato, Ipomoea batatas (L.) Lam", Biotech Lett, 27, pp.1841~1845 (2005)
- Krysan PJ, "T-DNA as an insertional mutagen in Arabidopsis", Plant Cell, 11, pp.2283~2290 (1999)
- Kuiper HA, "Assessment of the food safety issues related to genetically modified foods", Plant J, 6, pp.503~528 (2001)
- Lee YH, "Transgenic peppers that are highly tolerant to a new CMV pathotype", Plant Cell Rep, 28, pp.223~232 (2009)
- Mannerliöf M, "Screening of transgenic plants by multiplex", Plant Mol Biol. Rep., 15, pp.38~45 (1997)
- Matsuoka T, "A multiplex PCR method of detecting recombinant DNAs from five lines of genetically modified maize", J Food Hyg Soc Jpn, 42, pp.24~32 (2001)
- Yamaguchi H, "Two detection methods of genetically modified maize and the state of its import into Japan", Food Control, 14, pp.201~206 (2003)