Detection and Expression Analysis of Neomycin Phosphotransferase ⅡGene (NPTⅡ) in Muscle of Transgenic Tanichthys albonubes Expressing the Red Fluorescent Protein
Abstract：For ornamental purpose, our laboratory chose fertilized egg of Tanichthys albonubes used as a receptor to transfer red fluorescent protein vector of sea anemone in 2003. Transgenic T. albonubes expressing the red fluorescent protein was obtained. This paper aimed at evaluating its biological safety by analyzing expression and existence time of neomycin phosphotransferase Ⅱgene(NPTⅡ). PCR amplification and rapid diagnosis strip were applied on selectable marker gene detection and NPTⅡexpress detection in transgenic T. albonubes and non-transgenic T. albonubes. The results showed that NPTⅡ gene and protein product were detected in transgenic T. albonubes, but that were not detected in non-transgenic T. albonubes. To quantify the expression of NPTⅡ protein in muscle of transgenic T. albonubes, enzyme linked immunosorbent assay (ELISA) was performed at different death time in natural water (water temperature at 20~25℃). The death times are 0(fresh muscle), 24, 48, 72, 96, 120 and 144 h group, respectively. The result showed that the NPTⅡ protein content in the fresh muscle of transgenic T. albonubes was 9.12 ng/g, and the amount of NPTⅡ protein in the muscle of transgenic T. albonubes was decreased with death time prolonging. At the NPTⅡ protein content of the muscle was close to zero at 96 h after death . The results indicate that the NPTⅡprotein in transgenic T. albonubes muscle is so easy to be degraded under nature conditions and we speculate that it has low security risk to environment.
