Study On Agrobacterium-mediated Transformation Of Anthocyanin Regulatory Gene Lc-NtAn2into Tobacco

Anthocynins is the important secondary metabolites in plants, and it not only plays animportant role on plant growth and reproducton, disease resistance and stress resistance, butalso the prevention and treatment of human diseases. The regulation of anthocyninsbiosynthesis is mainly the transcriptional control of structural gene by the regulatory factors,and there are three types of transcription factor including R2R3-MYB, bHLH and WD40protein involved in the regulation of anthocynins biosynthesis. In addition to the interaction ofinternal factors (structural genes and transcription factor) in plant itself, the induced factors ofplant anthocyanin synthesis also include various kinds of environmental factors such as light,temperature, sugar and others. Studies on anthocyanin regulatory factors not only contributeto the improvement of crop quality, but also can help people with high nutritional value andhealth care value food. The NtAN2gene that encodes a R2R3-MYB transcription factor,interacts with the BHLH transcription factor in tobacco to regulate anthocynins biosynthesisin ordinary tobacco flowers; Lc gene encodes a bHLH transcription factor involved inanthocyanin biosynthesis in maize and regulate the color of many tissues such as leaf vein,leaf ligule, leaf margin, glumes and so on. In this study, with the leave of ordinary tobaccovariety "97204" as transformation receptor, Agrobacterium-mediated genetic transformationoptimized regeneration system has been established, and the Lc-NtAn2gene has beentranslated into tobacco variety "97204" to study the effect of Lc-NtAn2gene on anthocyaninsynthesis in tobacco. The main results are as follows:1. The leaf regeneration system of ordinary tobacco variety "97204" has been optimized.The best concentration ratio of6-BA/NAA inducing shoot regeneration in tobaccodifferentiation medium is1.0/0.1(mg/L).2. The technique system of ordinary tobacco variety "97204" of the Lc-NtAn2genemediated by Agrobacterium has been established. The suitable hygromycin concentration ofselecting transgenic tobacco plants is10mg/L; the suitable preculture time of the tobaccoleaves is2d; the highest frequency is observed when explants are immersed in bacteriumsuspension at OD600of0.5for5~8minutes; the suitable co-culture time of tobacco leaves andagrobacterium is2d; Cef should be used as the bacteriostatic antibiotics in the selection anddifferentiation medium, and its optimum working concentration is500mg/L. 3. PCR and RT-PCR analysis demonstrated that Lc-NtAn2gene has been integrated intoplant genome and could transcript normally; determination of anthocyanin content in leavesof five transgenic plants showed that the anthocyanin content in leaves of four transgenicplants increased, and one decreased.