Determination Of Transgene Copy Number Based On SAQPCR And Functional Analysis Of MrMYB1Based On Transformation

Quantitative real-time polymerase chain reaction (qPCR) has been previously applied to estimate transgene copy number in transgenic plants. However, the results can be erroneous owing to inaccurate estimation of PCR efficiency. Here, a novel qPCR approach, named standard addition qPCR (SAQPCR), was devised to accurately determine transgene copy number without the necessity of obtaining PCR efficiency data. The procedures and the mathematical basis for the approach are described. A recombinant plasmid harboring both the internal reference gene and the integrated target gene was constructed to serve as the standard DNA. It was found that addition of suitable amounts of standard DNA to test samples did not affect PCR efficiency, and the guidance for selection of suitable cycle numbers for analysis was established. Samples from six individual To tomato(Solanum lycopersicum Mill.) plants were analyzed by SAQPCR, and the results confirmed by Southern blot analysis. The approach produced accurate results and required only small amounts of plant tissue. It can be generally applied to analysis of different plants and transgenes. In addition, it can also be applied to zygosity analysis.MrMYB1, an R2R3MYB transcription factor (TF) gene associated with anthocyanin biosynthesis in Chinese bayberry(Myrica rubra Sieb. and Zucc), was introduced into Arabidopsis, tobacco (Nicotiana tabacum) and tomato under the control of the cauliflower mosaic virus35S promoter. The overexpression of MrMYBl induced anthocyanin accumulation in all tissues of Arabidopsis and but tobacco leaves did not become anthocyanic. The anthocyanin biosynthetic pathway, including chalcone synthase (CHS), dihydroflavonol4-reductase (DFR) and anthocyanidin synthase (ANS), and the bHLH transcriptional partner TRANSPARENT TESTA8(TT8), were up-regulated significantly in MrMYB1-overexpressing Arabidopsis. In MrMYB1-overexpressing tobacco petals, ovaries and young seeds, anthocyanin biosynthetic genes and bHLH partners NtAnla and NtAnlb, were up-regulated. In contrast, high expression of MrMYBl in transgenic tobacco leaves did not induce the expression of anthocyanin biosynthesis. Unlike in petals and ovaries, the foliar transcript level of NtAn1a and NtAn1b, was extremely low and not stimulated by MrMYB1transformation. In MrMKB1-overexpressing tomato leaves, petals, flesh and seeds, anthocyanin biosynthetic genes were not affected except SIDFR and bHLH partners were up-regulated slightly only in the seeds was a little higher. These results show that higher expression of an endogenous bHLH partner, either intrinsically or stimulated by exogenous gene transformation, is required for anthocyanin production plant tissues.