Functional Characterization Of OsMADS15and Screening Its Interactors By Yeast Two Hybrid System

Plant development and architecture is a complicated process in which series of genes expressed spatio-temporally. Among the genes, transcription factors play important roles in regulating genes expression in the right time and right space. Plant MADS-box gene family functions in many physiological processes such as plant development, metabolism, response to biotic and abiotic stimulus.Rice OsMADS15is a member of AP1/SQUA subfamily. In this study, we constructed a overexpression cassette of OsMADS15, and delivered to rice callus by agrobacterium-mediated transformation. Semi-quantitative PCR indicated the OsMADS15of wild type rice expresses in node, internode, mature leaf, palea and lemma, lodicule, but is absent or in low aboundance in callus, root, stamen and carpel. In the OsMADS15overexpression line, the gene is ectopically expressed in high aboundance. Precocious phenotypes were observed when rice plants expressing OsMADS15, and plants were dwarfism. In the seedling stage of the OsMADS15transgenic plants, the culms consisted of a series of elongated internodes and nodes. On the elongated nodes of the precocious culms were the flourishing shoot-borne crown roots. Real Time PCR indicated the WOX11was up-regulated by ten times in transgenic plants. In addition, the transgenic rice plants tillered later and less than the non-transgenic plants in the tillering stage. Another distinc phenotype is the transgenic plants had more elongated nodes. We also discerned the differentially expressed protein spots from panicle or mature leaf of Wt and transgenic rice using2-DE.A cDNA library of rice whole plant was constructed. The estimated titer of the library was1.8×106cfu/ml. PCR amplification of the insertions indicated the lengths of library were mostly larger than750bp.We screened proteins interacting with OsMADS15in the rice cDNA library using yeast two hybrid system. After selection, we totally obtained86His+Ade+LacZ+transformants. After sequencing and removing the repeating genes, we finally got55UniGenes. We further conducted the GO (Gene Ontology) annotation to functionally analyze the identified interactors. The genes were classified into three GO categories:cellular component, molecular function and biological process. In the cellular component category, proteins mapping to cell, cell part, organelle and macromolecular complex related GO terms were the most abundant. In the molecular function category, most proteins were addressed to terms of binding and catalytic activity. Considering the biological process category, most of proteins were involved in the metabolic process and cellular process. We identified three MADS-box genes as well: OsMADS1, OsMADS8and OsMADS14. OsMADS1and OsMADS8are E-class genes, while OsMADS14is an AP1/SQUA subfamily gene. BIFC assay was applied to confirm the interactions in vivo between OsMADS15and three candidate interactors (OsMADS1, OsMADS14、OsMADS8) in tobacco epidermis cells. The results showed that the nYFP (which linked to OsMADS15) and cYFP (which linked to OsMADS1、OsMADS14、OsMADS8respectively) could complement to emit fluorescence in the nucleus, indicating the proteins they fused to can interact with each other in the plant cells. They might function in quartet complexes with OsMADS15. This studies laid foundations for further researching the functions of OsMADS15in rice flower development and vegetative organs growth and development in future.