Improvement And Application Of Real-time Quantitative PCR-based Transgene Detection Techniques For Plants

Transgene detection is an important step for transgenic research. In this study, we used Micro-Tom and Kumquat as plant materials, developed a rapid DNA preparation protocol for real-time quantitative PCR analysis to detect transgenic plants and a recombinant plasmid system for screening the optimal primers. Using an optimized real-time quantitative PCR system, the transgene copy number of transgenic kumquat plants was confirmed. The results are summarized below:(1) A protocol for rapid preparation of DNA to detect transgenic plants with real-time quantitative PCRA simple and rapid genomic DNA preparation protocol for tomato leaves was presented, which is suitable for real time quantitative PCR analysis. Individual genomic DNA could be obtained from twelve samples within 30 minutes. Successful target DNA amplification was obtained in a 12.5μL PCR system, in which genomic DNA was extracted from leaf size ranging from 2 to 50 mm2. And genomic DNA varying from 0.1 to 0.2μL was used successfully as PCR template. Two pairs of primers, corresponding to ELIP gene (a plant internal gene) and HPT gene (the selectable marker of the transgene) were used for real time PCR amplification. The transgene copy number in transgenic tomato plants can be determined approximately according toΔCt between the transgene and the endogenous gene.(2) Construction of recombinant plasmid for screening the best primers.Recombinant plasmid HPT-ELIP, containing an internal tomato gene ELIP and the selectable marker gene, was constructed. Optimal primer pairs P438+P439 and SP061+SP062 were chosen for real-time quantitative PCR. Recombinant plasmid CS-NPTII, containing an internal gene kumquat CS and the target gene NPTII, was also constructed, and optimal primer pairs CS001+CS002 and SP069+SP070 were chosen, which was used in the next step for accurate detennination of transgene copy number. (3) Establishment of a real-time quantitative PCR system with additive recombinant plasmid to identity transgene copy number in transgenic plants.Using the optimized primers for the kumquat CS gene (internal reference gene) and NPTII gene (transgene), respectively, a real-time quantitative PCR method with additive recombinant plasmid to determine transgene copy number was established. The copy number of the transgene in transgenic kumquat obtained from this approach was consistent with previous Southern blot results.