Abstract:

【Objective】 Screening the interacting protein of WDR40 protein TRANSPARENT TESTA GLABRA 1（TTG1）by constructing the cDNA library of Chinese cabbage seeds, the molecular mechanism of TTG1 in the regulation of the formation of MBW ternary complex will be explored.【Method】 Seeds of brown-seeded Chinese cabbage ‘B147’ were used as materials for RNA extraction and cDNA library construction. The bait vector pGBKT7-TTG1 was constructed by gateway technology and then yeast two-hybrid screening were performed.【Result】 The library capacity was 1.2×10⁷ CFU and library titer was 5.0×10⁷ CFU/mL with average length of the insert greater than 1 000 bp. The bait vector had no self-activating activity in yeast. A total of 38 positive interacting proteins were obtained by hybridizing the bait vector pGBKT7-TTG1 with the cDNA library. Function prediction revealed one of the proteins annotated as a MYB73 transcription factor. Sequence analysis results showed that this gene contained R2R3-MYB type suppressor conserved motifs C1 and C2, speculated that this gene may be the R2R3-MYB suppressor involving in the seed coat color formation in Chinese cabbage, suggesting that there may be different MYB transcription factors in the regulation network affecting the formation of proanthocyanidins.【Conclusion】 In this study, a yeast two-hybrid cDNA library of Chinese cabbage seed-specific tissue is constructed and a total of 38 TTG1-positive interacting proteins are obtained. We found the R2R3-MYB type suppressor MYB73 that may affect the formation of proanthocyanidin color in cabbage seed coat for the first time. This study lays a good foundation for exploring the regulatory network of proanthocyanindins formation.

Key words: Chinese cabbage seed coat color, cDNA library, yeast two-hybrid, interactive protein, MYB73, gene clone, expression analysis