Construction of Yeast Two-hybrid Bait Vector and the Screening of Proteins Interacting with HbICE1 in Hevea brasiliensis
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摘要: 寒害是我国植胶区的主要自然灾害之一,不仅影响橡胶产量还威胁到橡胶树的生存。拟南芥中,ICE1是低温胁迫信号通路中的重要转录激活因子,但橡胶树冷胁迫信号途径中包括HbICE1在内的大部分关键基因尚未被克隆与鉴定,与HbICE1蛋白发生互作的蛋白也不清楚,成为严重阻碍橡胶树抗寒分子机理研究的瓶颈。为了筛选和鉴定与巴西橡胶树HbICE1 蛋白发生互作的蛋白,阐明橡胶树抵御寒害胁迫的分子机理,本文首先通过PCR扩增出HbICE1基因的编码序列(约1400 bp),然后将目标序列插入pGBKT-7载体,构建酵母双杂交诱饵载体;将该载体转入酵母Y2H菌株感受态细胞中,并在缺陷型培养基上检测其自激活活性,发现HbICE1基因的自转录激活在含有30 mmol/L和更高浓度3-AT(3-氨基-1,2,4-三氮唑)的培养基上能得到有效抑制;进一步通过与橡胶树cDNA文库进行酵母双杂交,筛选出一些可能与HbICE1发生互作的蛋白,包括DNA结合蛋白、核糖体蛋白和功能未知蛋白。本研究结果为橡胶树抗寒机理研究提供了重要理论依据。Abstract: Cold stress is a major issue in the rubber plantation areas of China. It not only affects rubber production, but also threatens the survival of the rubber tree. ICE1 is a transcriptional activator in the cold-response pathway of Arabidopsis thaliana L.. However, most key genes, including HbICE1, in the cold-response pathways of Hevea brasiliensis (Willd. ex A. Juss.) Muell. have not yet been cloned, and the proteins that interact with HbICE1 are unclear, which has hampered the advance of molecular mechanism research of cold tolerance in H. brasiliensis. We cloned HbICE1 and constructed its bait vector, and then transformed pGBKT-7-HbICE1 into yeast Y2H strain. The transformed yeast cells were plated on selection medium for bait auto-activation test. The auto-activation of HbICE1 was inhibited on selection medium (SD/-Trp/-His/-Ade) supplemented with 30 mmol/L 3-AT (3-amino-1,2,4-triazole) and higher concentrations. Thereafter, the proteins that interacted with HbICE1 were screened through the yeast two-hybrid method, which included DNA-binding protein, ribosomal protein and function unknown proteins. This study provides an important theoretical basis for the molecular mechanism of the rubber tree’s response to cold stress.
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Keywords:
- Hevea brasiliensis /
- HbICE1 /
- Yeast two-hybrid /
- Proteins interaction /
- Chilling tole-rance
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