Construction of yeast one-hybrid library and screening of transcription factors regulating LhMYBSPLATTER expression in Asiatic hybrid lilies (Lilium spp.)

Background Anthocyanins, which belong to flavonoids, are widely colored among red-purple pigments in the Asiatic hybrid lilies (Lilium spp.). Transcription factor (TF) LhMYBSPLATTER (formerly known as LhMYB12-Lat), identified as the major kernel protein, regulating the anthocyanin biosynthesis pathway in 'Tiny Padhye' of Tango Series cultivars, which the pigmentation density is high in the lower half of tepals and this patterning is of exceptional ornamental value. However, the research on mechanism of regulating the spatial and temporal expression differences of LhMYBSPLATTER, which belongs to the R2R3-MYB subfamily, is still not well established. To explore the molecular mechanism of directly related regulatory proteins of LhMYBSPLATTER in the anthocyanin pigmentation, the yeast one-hybrid (Y1H) cDNA library was constructed and characterized. Results In this study, we describe a yeast one-hybrid library to screen transcription factors that regulate LhMYBSPLATTER gene expression in Lilium, with the library recombinant efficiency of over 98%. The lengths of inserted fragments ranged from 400 to 2000 bp, and the library capacity reached 1.6 x 10(6) CFU of cDNA insert, which is suitable to fulfill subsequent screening. Finally, seven prey proteins, including BTF3, MYB4, IAA6-like, ERF4, ARR1, ERF WIN1-like, and ERF061 were screened by the recombinant bait plasmid and verified by interaction with the LhMYBSPLATTER promoter. Among them, ERFs, AUX/IAA, and BTF3 may participate in the negative regulation of the anthocyanin biosynthesis pathway in Lilium. Conclusion A yeast one-hybrid library of lily was successfully constructed in the tepals for the first time. Seven candidate TFs of LhMYBSPLATTER were screened, which may provide a theoretical basis for the study of floral pigmentation.