Cloning Of Anthocyanin Regulatory Genes From Turnip 'Tsuda' And Its Transformation To Chrysanthemum Morifolium

In vivo, the biosynthesis of anthocyanins from its beginning phenylalanine is a series of enzyme-catalyzed reactions. The structural genes encoding these enzymes are regulated by transcriptors encoded by regulatory genes, for their spatial and temporal expression, to regulate the anthocyanins biosynthesis. The transcriptor families regulating anthocyanin biosynthesis have been identified include:WRKY, Zn-finger, BZIP, MADS-box, MYB, bHLH, WD40 etc.. Studies on Arabidopsis, petunia, maize reveal the late pathway of anthocyanins biosynthesis is regulated by a complex of MYB, bHLH and WD40. Antibiotic selection system is widely utilized in the transformation of chrysanthemum (Dendranthema×gandiflorum), as while few is reported utilizing sugar selection. In this study, We selected turnip 'Tsuda'(Brassica rapa L. 'Tsuda') as the test material, late genes were cloned and their expression levels were studied. Also, we transformed the cloned gene PAP1 into Chrysanthemum 'flame' by both antibiotic and sugar selection system.Four late genes of anthocyanin pathway were cloned from turnip 'Tsuda' by RT-PCR or RACE (rapid amplification cDNA end), including MYB-like PAP1, bHLH-like EGL3/TT8, WD40-like TTG1. Analysis on these genes by informatics methods reveals that PAP1 is a R2R3-MYB protein with SANT superfamily conserved domains containing DNA binding site; EGL3 and TT8 contain HLH superfamily conserved domain with DNA binding region, E-box specificity site, dimerization interface; TTGl contains WD40 superfamily conserved domain.Utilizing 3 month old turnip 'Tsuda', by Real-time PCR we analyzed the expression levels of cloned genes in different tissues and in white epidermis of swollen hypocotyls induced by UV-A light. The results reveal that all the four genes are highly expressed in red root epidermis where anthocyanins are synthesized. Their expression levels become higher with the induction of UV-A light, while TTG1 is not so obvious. All these indicate that the four genes function in the biosynthesis of anthocyanins.Through 'Gateway' technology PAP1 was subcloned into three different expression vector: pH7WG2D, pCAMBIA-1301-PMI, pCAMBIA-1301-XYL, the selection agents corresponding to which are hygomycin, mannose and xylose when transforming chrysanthemum. PAP1 was then transformed into chrysanthemum by Agrobacterium-mediated method. We got four transgenic plants by hygomycin selection system and two by mannose selection system after PCR identification. Compared with xylose, mannose is poisonous to plant cells, so it can effectively reduce the false-positive shoots formation and therefore is more suitable as a selection agent in chrysanthemum transformation.