Estimating Transgene Copy Number In Citrus By Real-Time Quantitative PCR

Transgene copy number has a great impact on the expression level and stability of exogenous gene in transgenic plants, so transgene copy number analysis is identified as one most important task after obtaining transgenic plants. In this paper, TaqMan real-time PCR was used to estimate the copy number of exogenous genes in transgenic precocious trifoliate orange obtained previously, in order to overcome the limitations of Southern blot analysis, which is labor-intensive, time-consuming, in considerable needs of DNA, etc. This system was analyzed 26 MAC12.2 transgenic precocious trifoliate oranges and invited a conserved endogenous reference gene coding for lopid transfer protein (PtLTP) We developed a real-time PCR assay which permitted the determination of the copy number of transgene (MAC12.2 and NPTâ…¡), relative to PtLTP in transgenic citrus lines. This analysis was proved high-throughput and effective, and it could be performed at much earlier stages in slow-growing woody plants. The main results are as follows:1.26 PCR positive plants were selected for further Southern blot analysis, which could confirm the stable integration of the MAC12.2 gene cassette. Different integration patterns from one to five copies at different loci were observed with Hindâ…¢.2. PtLTP gene were amplified in precocious trifoliate orange (Poncirus trifoliate L. Raf) based on the sequence of LTP gene from sweet orange (Citrus sinensis L. Osbeck). This gene was used as a reference gene.3. Three standard curves (one for endogenous gene LTP and others for transgenic genes) were obtained. We estimated transgene copy number by TaqMan quantitative real-time PCR. There were four lines (P3, P13, P22, P23), accounting for 15.4% of 26 tested lines, in which the MAC12.2 copy number was not parallel with the NPTâ…¡copy number, that suggested possible T-DNA rearranged during the process of chromosomal integration.4. To confirm the feasibility of the method by real-time PCR to analyze transgene copy number in depth, first we compared copy numbers of MAC 12.2 derived from real-time PCR and Southern blot results. In summary, the correlation coefficient between Southern blot and TaqMan assay data is rather good (92.21%), only two samples (P20 and P22) showed less correlation between both results. Furthermore, the copy number in all the tested lines were sufficiently at integer loci and the variation of the copy number was minimal in the range of 0.07-0.12. Apparently, the result conformed to true value. "P" and "SS" two parameters revealed that the feasibility and the accuracy were high. P fell in the range of 1.3%-22.5%. SS showed from 0.004 to 0.058.