蛋白间的相互作用对于生物学功能的发挥是非常重要的。MA DS-box基因的功能主要通过形成同源或异源二聚体或四聚体来实现。水稻OsMA DS15是一个AP1/SQUA亚家族的成员，在水稻开花时间调控、不定根的发育、茎的生长和内稃的发育等过程中具有重要作用，但与之有相互作用的蛋白却知之甚少。本实验用酵母双杂交法从水稻(Oryza sativa ssp。japonica)cDNA文库中筛选与OsMA DS15有相互作用的蛋白。构建了pGB KT7-OsMA DS15诱饵表达载体，然后与pGADT7-cDNA library共转化到酵母(Saccharomyces cerevisiae)AH109中，通过顺序筛选HIS3、A DE2和LacZ3个报告基因的表达，共得到25个UniGene。在烟草(Nicotiana benthamiana)细胞中用BIFC实验验证OsMADS15和候选基因OsMA DS1、OsMA DS14、Os MA DS8之间的相互作用，结果表明在烟草细胞的核内，OsMADS15相连的nYFP可以与OsMA DS1、OsMA DS14、Os MA DS8相连的cYFP分别形成互补而产生荧光，证实其间在植物体内也具有相互作用。本实验为今后近一步研究OsMADS15在水稻花发育和水稻营养器官生长发育中的功能提供了基础资料。
Screening of Rice OsMADS15 Interacting Proteins by Yeast Two Hybrid System
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 0sMADS15 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 nce(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) AH 109. 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: OsMADSl, psMADS8 and psMADS14. OsMADSl and psMADS8 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, psMADS8 and OsMADS14) in tobacco(Nicotiana benthamiana) epidermal leaf cells. The results showed the Nyfp (which linked to OsMADS15) and Cyfp (which linked to OsMADS1, psMADS14 and psMADS8, 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.
Rice, psMADS15, Yeast two hybrid, Cdna library, BIFC
国家科技期刊平台
NSTL
万方数据
维普
