Characterization of the ZmbHLH122 transcription factor and its potential collaborators in maize male reproduction
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OsEAT1 (DTD), a stamen-specific bHLH transcription factor, is a master regulator in rice male reproduction. To elucidate its functional mechanism in maize male reproduction, the maize orthologue ZmbHLH122 of OsEAT1 was isolated in this study. Sequence analysis revealed that the ZmbHLH122 coding sequence consists of 1422 bp which encode a protein of 473 amino acids. Bioinformatic analysis confirmed that ZmbHLH122 belongs to the bHLH TF family. The subcellular localization of ZmbHLH122-eGFP in rice protoplasts showed that ZmbHLH122 is a nuclear-localized protein. Yeast one-hybrid assays demonstrated that ZmbHLH122 exhibits weak transactivation ability. Genome-wide coexpression analysis identified 751 potential collaborators of ZmbHLH122, most of which are highly expressed in maize anthers and tassels, and sequence alignment analysis revealed that 43 coexpressed genes are homologous to Arabidopsis male fertility-related genes. Expression pattern analysis confirmed that ZmbHLH122 and its potential collaborators Ms23 (ZmbHLH16), ZmbHLH51, ZmbHLH66 (Ms32) and ZmMYB74 are preferentially expressed in the maize male reproductive organs, especially those at the uninucleate and early binucleate stages. Yeast two-hybrid assays revealed that ZmMYB74 directly interacts with two maize stamen development regulators, Ms23 (ZmbHLH16) and ZmbHLH51, and indirectly collaborates with ZmbHLH122. Our study lays the foundation for further understanding of the ZmbHLH122 molecular function in maize male reproduction.
KeywordsbHLH transcription factor Yeast hybrids Coexpression analysis Male reproduction Maize
MC and YL designed the research and wrote the article. YL performed most of the experiments. GW, ZZ, and PZ performed some of the experiments. HL, SZ, and CL assisted in the data analysis. All authors have read and approved the final manuscript.
This work was supported by Grants from the National Key Research and Development Program of China (No. 2016YFD0101206) and the Platform for Mutation Breeding by Radiation in Sichuan (No. 2016NZ0106).
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Conflict of interest
The authors declare that they have no competing interests.
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