Construction and Application of a Plasmid Reference Molecule for Detecting Transgenic Rice

Abstract

Abstract: Five targets, including the CaMV35S promoter, NOS terminator, Bar gene, HPT gene and NPTⅡ gene were selected to be detecting targets of transgenic rice screening, based on the application frequency of the regulation elements and function genes used in rice（Oryza sativa）genetic engineering research. A rice reference gene SPS, five screening targets（CaMV35S promoter, NOS terminator, Bar gene, HPT gene and NPTⅡ gene）and three event-specific detection fragments（TT51-1, KF-6, KMD1）were fused using a overlap extension PCR technique and cloned into a plasmid vector pBluescript II SK+ to construct pBS Rice. The results showed that the plasmid molecules pBS Rice could be applied not only for the screening of genetically modified rice, but also in the event-specific detections of insect-resistant rice TT51-1, KF-6 and KMD1. This study provides a raw materials independent positive control with a coverage rate of 100% for the worldwide approved rice events, meeting the needs of the transgenic rice security supervision.

Key words: Transgenic rice, Reference plasmid molecule, Qualitative PCR, Screening detection, Event-specific detection

Li Xiaofei,Tan Xiaoli,Li Jun,Wu Yuhua,Cao Yinglong. Construction and Application of a Plasmid Reference Molecule for Detecting Transgenic Rice[J]. Biotechnology Bulletin, 2013, 0(7): 71-77.

References

[1] Toriyama K, Arimotoa Y, Uchimiyaa H, et al. Transgenic rice plants after direct gene transfer into protoplasts [J]. Nature Biotechnology, 1988, 6：1072-1074.
[2] Zhang HM, Yang H, Rech EL, et al. Transgenic rice plants produced by electroporation-mediated plasmid uptake into protoplasts [J]. Plant Cell Reports, 1988, 7：379-384.
[3] Zhang W, Wu R. Efficient regeneration of transgenic plants from rice protoplasts and correctly regulated expression of the foreign gene in the plants [J]. Theor Appl Genet, 1988, 76：835-840.
[4] James C. Global Status of Commercia-lized Biotech/GM Crops [M]. Ibercaja, Spain：The International Service for the Acquisition of Agribiotech Applications, 2012.
[5] Shu QY, Ye GY, Cui HR, et al. Transgenic rice plants with a synthetic cry1Ab gene from Bacillus thuringiensis were highly resistant to eight lepidopteran rice pest species [J]. Molecular Breeding, 2000, 6：433-439.
[6] Lu CM. The first approved transgenic rice in China [J]. Genetically Modified Crops, 2010, 1：113-115.
[7] Tu J, Datta K, Alam MF, et al. Expression and function of a hybrid Bt toxin gene in transgenic rice conferring resistance to insect pests [J]. Plant Biotechnology, 1998, 15（4）：195-203.
[8] Tu J, Zhang GA, Datta K, et al. Field performance of transgenic elite commercial hybrid rice expressing Bacillus thuringiensis dendotoxin [J]. Nature Biotechnology, 2000, 18：1101-1104.
[9] Tu J, Datta K, Oliva N, et al. Site-independently integrated transge-nes in the elite restorer rice line Minghui 63 allow removal of a selec-table marker from the gene of interest by self-segregation [J]. Plant Biotechnology Journal, 2003, 1：155-165.
[10] 舒庆尧, 叶恭银, 崔海瑞, 等. 转基因水稻”克螟稻”选育[J].浙江农业大学学报, 1998, 24（6）：579-580.
[11] Rong J, Song ZP, Su J, et al. Low frequencies of transgene flow between Bt/CpTI rice and their nontransgenic counterparts under alternating cultivation [J]. Biodivers Sci, 2006, 14：309-314.
[12] 李冬虎, 傅强, 王锋, 等.转sck/cry1Ac双基因抗虫水稻对二化螟和稻纵卷叶螟的抗虫效果[J].中国水稻科学, 2004, 18（1）：43-47.
[13] 赵红盈, 张永军, 吴孔明, 等.转cryAc/CpTI双价抗虫水稻cry1Ac杀虫蛋白的表达特性及其对二化螟的毒杀效果[J].农业生物技术学报, 2004, 12：76-79.
[14] Marta H, Adolfo R, Teresa E, et al. A rapeseed-specific gene, acetyl-CoA carboxylase, can be used as a reference for qualitative and Real-Time quantitative PCR detection of transgenes from mixed food samples [J]. Journal of Agricultural and Food Chemistry, 2001, 49（8）：3622-3627.
[15] 李飞武, 李葱葱, 邢珍娟, 等. 抗草甘膦系事件特异性定量PCR检测方法的建立[J].大豆科学, 2009, 28（2）：296-300.
[16] Lievens A, Bellocchi G, De BD, et al. Use of pJANUS02-001 as a calibrator plasmid for Roundup Ready soybean event GTS40-3-2 detection：an interlaboratory trial assessment [J]. Analytical and Bioanalytical Chemistry, 2010, 396（6）：2165-2173.
[17] Yang LT, Pan AH, Zhang KW, et al. Qualitative and quantitative PCR methods for event-specific detection of genetically modified cotton Mon1445 and Mon531 [J]. Transgenic Research, 2005, 14（6）：817-831.
[18] Hernandez M, Pla M, Esteve T, et al. A specific real-time quantitative PCR detection system for event MON810 in maize Yield Gard based on the 3￠-transgene integration sequence [J]. Transgenic Research, 2003, 12（2）：179-189.
[19] Lee SH, Min DM, Kim JK. Qualitative and quantitative polymerase chain reaction analysis for genetically modified maize MON863 [J]. Journal of Agricultural and Food Chemistry, 2006, 54：1124-1129.
[20] Nielsen CR, Berdal KG, Holst-Jensen A. Characterization of the 5 integration site and development of an event-specific real-time PCR assay for NK603 maize from a low starting copy number [J]. European Food Research and Technology, 2004, 219：421-427.
[21] Made D, Degner C, Grohmann L. Detection of genetically modified rice：a construct-specific real-time PCR method based on DNA sequences from transgenic Bt rice [J]. European food Research and Technology, 2006, 224：271-278.
[22] Wu G, Wu YH, Nie SJ, et al. Real-time PCR method for detection of the transgenic rice event TT51-1 [J]. Food Chemistry, 2010, 119（1）：417-422.