The Study Of A Maize Transcription Factor ZmMADS47 Regulating Zein Gene Transcription Through Interacting With Opaque2

Opaque2 (O2) is an important bZIP transcription factor in the development of maize kernels (Zea mays). Studies have shown that O2 can widely participate in the development of kernels and mainly regulates the storage protein-zein which has the largest contents in a kernel.O2 can interact with multiple transcription factors as well as cytoskeletal proteins and influence their biological functions. This research aims to analyze the biological function of ZmMADS47, a new O2-interacting protein.Yeast two-hybrid screen was adopted in the preliminary study and multiple putative O2-interacting proteins were gained including ZmMADS47, a MADS-box transcription factor. In the study, we used co-immunoprecipitation experiment and GST Pull-down experiment and testified the interaction between ZmMADS47 and O2. Gene expression analysis found that ZmMADS47 could express in multiple tissues. Gene phylogenetic tree analysis suggests that ZmMADS47 is an homologous to OsMADS47 of rice. The detection on the transcription factor feature of ZmMADS47 including nuclear localization and transcriptional activation ability found that the N terminal and the full-length of ZmMADS47 had nuclear localization ability; the C terminal of it had obvious transactivation ability while the full-length protein had a low transactivation ability.Transgenic RNAi knockdown of ZmMADS47 expression was gained through transgenic maize. The analysis of RNA-seq, transmission electron microscope and biochemical components in RNAi transgenic kernels suggested that the low expression of ZmMADS47 made the expression amount of α-zein and 50-kD γ-zein decreased in 15DAP and some protein bodies became smaller and even irregular. DNA-protein interaction experiment in vitro affirmed that ZmMADS47 was directly interacted with the CATGT sequences of α-zein and 50-kD γ-zein promoters. Transcriptional activation experiment on onion epidermal cells affirmed that ZmMADS47 cannot activate the transcription of the target promoter independently while after interacting with O2, it can strengthen the transcription of the target promoter with different degrees of strength. The analysis of deletion mutation in O2 protein transcriptional activation structural domain and 50-kD γ-zein promoter CATGT sequences as well as the deletion of the seven amino acids in ZmMADS47 C terminal suggested that the protein interaction between 02 and ZmMADS47 may change the conformation of ZmMADS47 and release the transcriptional activation ability in the transcriptional activation domain in the C terminal of ZmMADS47.Through this study, ZmMADS47, a new 02-interacting protein, has been affirmed and it is discovered that it is a new zein gene transcription factor. ZmMADS47 conducts its function by a mechanism of interaction activation. The result of this research broadened our vision on the regulation of regulation of zein gene transcription.