Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, 100081, China.
BMC Plant Biol. 2021 Nov 29;21(1):563. doi: 10.1186/s12870-021-03347-1.
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.
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 × 10 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.
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.
花色苷属于类黄酮，广泛存在于亚洲杂交百合（百合属）的红紫色色素中。转录因子（TF）LhMYBSPLATTER（以前称为 LhMYB12-Lat）被鉴定为主要核蛋白，调节“Tango 系列品种‘Tiny Padhye’中的花色苷生物合成途径，该品种花瓣的下半部分色素密度较高，这种图案具有特殊的观赏价值。然而，对于属于 R2R3-MYB 亚家族的 LhMYBSPLATTER 的时空表达差异的调节机制的研究尚不完善。为了探索与 LhMYBSPLATTER 直接相关的调节蛋白的分子机制，构建并表征了酵母单杂交（Y1H）cDNA 文库。
本研究描述了一种用于筛选调控百合 LhMYBSPLATTER 基因表达的转录因子的酵母单杂交文库，文库重组效率超过 98%。插入片段的长度范围为 400 至 2000bp，文库容量达到 1.6×10CFU cDNA 插入物，适合进行后续筛选。最终，通过重组诱饵质粒筛选出包括 BTF3、MYB4、IAA6 样、ERF4、ARR1、ERF WIN1 样和 ERF061 在内的七个诱饵蛋白，并通过与 LhMYBSPLATTER 启动子的相互作用进行验证。其中，ERFs、AUX/IAA 和 BTF3 可能参与了百合花色苷生物合成途径的负调控。
首次成功构建了百合花瓣的酵母单杂交文库。筛选出 LhMYBSPLATTER 的七个候选 TF，为花色形成的研究提供了理论依据。
