|
|
|
| Screening of Rice OsMADS15 Interacting Proteins by Yeast Two Hybrid System |
| , , , , |
|
|
|
|
Abstract Interactions among proteins are important for the majority of biological functions. MADS-box genes exert their functions through formation of homo- or/and hetero- dimer/tetramer. Rice OsMADS15 is a member of APETALA1/SQUAMOSA(AP1/SQUA) subfamily, which plays important roles in biological processes such as flowering time regulation, adventitious roots development, stem growth and palea development. But little information was known for its interaction partners. In this study, we screened its interaction partners using yeast two hybrid system with a rice(Oryza sativa ssp. japonica) cDNA library. A bait plasmid pGBKT7-OsMADS15 was constructed, and because the OsMADS15 was predicted as a transcription factor, the bait plasmid pGBKT7-OsMADS15 was verified in advance that it did not autonomously activate the reporter gene HIS3. The bait plasmid and pGADT7-cDNA library were simultaneously transformed to yeast (Saccharomyces cerevisiae)AH109. After selection of HIS3, ADE2 and LacZ reporter genes subsequently agaist the positive (pCL1+BD and AD-T+BD-53) or negative controls (AD-T+BD-LamC), thirty-three positive colonies were obtained and the insertion cDNAs on pGADT7-cDNA library plasmids were PCR amplified and sequenced. Removing the redundant sequences, we finally got 25 unigenes. We identified three MADS-box genes as well: OsMADS1, OsMADS8 and OsMADS14. OsMADS1 and OsMADS8 were E-class genes, while OsMADS14 was an AP1/SQUA subfamily gene. BIFC assay was applied to confirm the interactions in vivo between OsMADS15 and the three candidate interactors (OsMADS1, OsMADS8 and OsMADS14) in tobacco(Nicotiana benthamiana) epidermal leaf cells. The results showed the nYFP (which linked to OsMADS15) and cYFP (which linked to OsMADS1, OsMADS14 and OsMADS8, respectively) could complement to emit fluorescence in the nucleus, indicating the fused proteins could interact with each other in the plant cells. The results might implicate that OsMADS15 function in tetramer complexes with OsMADS1, OsMADS8 and OsMADS14, as described in floral quartet model. These studies provide experimental data for further researching the functions of OsMADS15 in rice flower development and vegetative organs growth and development.
|
|
Received: 31 July 2012
|
|
|
|
|
|
|
