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利用已构建的月季分裂泛素酵母双杂文库,以月季乙烯受体蛋白RhETR3为诱饵,筛选能够与RhETR3结合的互作蛋白。经酵母回转验证共得到26个阳性克隆。在候选蛋白中发现一个TspO/MBR蛋白家族的成员,命名为RhTSPO1。通过RACE分离了RhTSPO1基因全长,共777 bp,开放阅读框(ORF)为579 bp,编码192个氨基酸。在酵母中,利用酵母生长和β–半乳糖苷酶活性(β-gal)验证了RhETR3和RhTSPO1之间确实存在互作。将RhETR3-GFP和RhTSPO1-mCherry融合蛋白共转入烟草,激光共聚焦显微镜观察发现RhTSPO1和RhETR3蛋白共同定位于内质网上。月季花朵自然开放过程中,RhTSPO1在花瓣中的表达呈现先增强后减弱的趋势,与开放和衰老过程紧密联系,表明RhTSPO1可能参与了花朵开放过程。本试验结果表明乙烯受体蛋白水平的互作调节可能也参与月季花朵开放和衰老的调节。
The double-stranded library of rose-splitting ubiquitin yeast was constructed and RhETR3 was used as bait to screen the interacting proteins capable of binding to RhETR3. A total of 26 positive clones were obtained by yeast rotation test. A member of the TspO / MBR family of proteins found in the candidate protein, named RhTSPO1. The full length of RhTSPO1 gene was isolated by RACE, which was 777 bp in length and 579 bp in open reading frame (ORF), encoding 192 amino acids. In yeast, the interaction between RhETR3 and RhTSPO1 was confirmed by yeast growth and β-galactosidase activity (β-gal). The RhETR3-GFP and RhTSPO1-mCherry fusion proteins were co-transfected into tobacco. Confocal laser scanning microscopy revealed that the RhTSPO1 and RhETR3 proteins co-localized on the endoplasmic reticulum. During the natural opening of rose flowers, the expression of RhTSPO1 in petals increased first and then decreased, closely linked with the process of opening and senescence, indicating that RhTSPO1 may be involved in the flower opening process. The results show that the interaction of ethylene receptor protein levels may also be involved in the opening of rose flowers and the regulation of aging.