Establishment of Real Time PCR Detection System of Expression level of Mi1.2 Transgenic Tomato Plant

[Objective] The research aimed to lay the foundation for the high-throughput screening for transgenic tomato plants.[Method] Total RNA was extracted from tomato leaves by CATB method to make Real Time PCR amplification.The detection system of the expression level of Mi 1.2 transgenic tomato plants were analyzed.[Result] The A260/A280 value of the extracted RNA was 1.78～1.88 and RNA had no obvious degradation.Under rigorous amplification conditions,the amplification efficiency of primer SYBR2 was higher than that of SYBR1.The suitable concn.of Mg2+ was 2.0 mg/L.The products of Real Time PCR amplification had the fine specificity.Its specific peak of the melting curve appeared at 84.5 ℃ nearby and there was extremely weak nonspecific peak at the point of slightly lower than 84.5 ℃ of the melting curve.The signal detection step in quantitative reaction should be set at the point of 84 ℃ The regression equitation obtained from Ct value of the amplification curve under the conditions of the molecular number of 4 kinds of different templates was Y=-3.78×log(copynumber)+ 39.50,with the correlation coefficient of 0.998.[Conclusion] The Real Time PCR system obtained from this test could be used for detecting the expression level of transgenic tomato plants.