Molecular analysis and quantitative detection of a transgenic rice line expressing a bifunctional fusion TPSP

The remarkable amount of public and scientific debate with regard to the safety of genetically modified (GM) crops has resulted in the implementation of mandatory risk assessments of newly developed GM crops, as well as mandatory labeling of GM crops and foods following their commercial release. Among the many currently available GM rice lines, transgenic rice lines that overexpress a trehalose-6-phosphate synthase/phosphatase (TPSP) fusion gene have attracted particular interest from researchers, by virtue of their superior tolerance to a variety of abiotic stresses. The primary objective of this study was to determine the copy numbers of the transgene element, and to identify the genomic sequences flanking the integration site of the transgene element in a selected TPSP transgenic rice line, in order to develop new event-specific detection techniques. The genomic sequences flanking the integration site of the transgene element were identified via an inverse PCR protocol. It was determined that a single copy of the TPSP fusion had been integrated into the transgenic line. An effort to find a rice sequence usable as an internal positive control for the screening of the transgenic rice resulted in the identification of an RBE4 gene sequence, which appears as one copy within the rice genome, and is rice-specific. The characterization of the genomic sequences flanking the transgene, as well as the availability of the internal positive control sequence, enabled the design of a qualitative PCR technique and a quantitative TaqMan-based detection method for this transgenic rice line. The results of this study may prove useful with regard to the large-scale screening of this transgenic rice line in the processes of risk assessment and commercialization, as well as in the development of novel methods for the detection of other transgenic rice lines.