【目的】构建ERF(ethylene-responsive element binding factor)转录因子基因W17的亚细胞定位载体和原核表达载体,验证W17是否具有核定位功能,阐明W17与GCC、DRE探针的体外结合特性,利用GUS瞬时表达系统分析W17蛋白的体内结合特性和转...【目的】构建ERF(ethylene-responsive element binding factor)转录因子基因W17的亚细胞定位载体和原核表达载体,验证W17是否具有核定位功能,阐明W17与GCC、DRE探针的体外结合特性,利用GUS瞬时表达系统分析W17蛋白的体内结合特性和转录激活功能,初步预测W17在植物胁迫信号传导途径中的作用。【方法】构建W17/163hGFP亚细胞定位载体,基因枪转化洋葱表皮细胞,暗培养24h后共聚焦显微镜下观察。构建W17/pGEX-4T-1原核表达载体,转入大肠杆菌BL21(DE3),IPTG(0.5mmol·L-1,3h)诱导,GST纯化柱纯化,纯化的融合蛋白与[γ-32P]ATP标记的GCC、DRE探针混合进行凝胶阻滞试验。构建GUS瞬时表达系统,通过农杆菌介导转化烟草,X-Gluc染色、酒精脱色后体视显微镜下观察。【结果】W17基因具有核定位功能,纯化的融合蛋白GST/W17能与正常GCC、DRE探针体外特异结合,与突变GCC、DRE探针不结合,在植物体内与GCC特异结合并能激活下游GUS基因表达。【结论】W17通过自身的NLS进入核内行使功能,参与了GCC-box调控的生物胁迫信号传导途径,还可能参与了非生物胁迫(盐胁迫)传导途径。展开更多
The study aims to detect the subcellular localization of ERF （ethylene-responsive element binding factor） transcription factor W17 protein, the interaction between W 17 and cis-acting regulatory elements GCC-box and...The study aims to detect the subcellular localization of ERF （ethylene-responsive element binding factor） transcription factor W17 protein, the interaction between W 17 and cis-acting regulatory elements GCC-box and DRE in vitro, the binding and transactivating ability in vivo, and the role of W17 in higher plant stress-signal pathway. Recombinant plasmid W17/163hGFP was introduced into onion epidermal cells by the particle bombardment method with a PDS 1000/He. Transformed cells were incubated for 24 h at 22℃ in the dark and green fluorescence was monitored under a confocal microscope. The gene W17 was fused N-terminus of GST （glutathione-S-transferase） in prokaryotic expression vector pGEX-4T-1 and then transformed into E. coli strain BL21 （DE3）. IPTG （0.5 mmol L-1） was added to induce the expression of recombinant GST/W17 for 3 h. The fused proteins were purified by GST purification columns, and then subjected to gel retardation assay with a 32p-labeled GCC or DRE sequence. The different reporter and effector plasmids were introduced into tobacco leaves through agroinfiltration, then transformed leaves stained by X-Gluc, faded with 75% alcohol and monitored under a Stereozooming microscope. The GFP fused with W17 protein was localized in the nuclei; SDS-PAGE assay demonstrated that the fused protein GST/W17 could be induced and purified with molecular weight at around 42.2 kD under the induction of IPTG. Purified fused protein was able to specifically bind to both the wild-type GCC-box and DRE element, but had no interaction with either the mutant DRE or GCC-box; W17 protein can bind to GCC-box and transactive downstream GUS gene in vivo. W17 can localize into the nuclei, and it may be involved not only in biotic stresses controlled by GCC-box, but also in abiotic stresses （e.g., salt-） induced signaling pathway.展开更多
The expression of Arabidopsis PDF1.2 gene is regulated by jasmonic acid (JA) and ethylene (ET). It also has been well documented that GCC box is an element re- sponsive to ET,however, the responsive mechanism of JA in...The expression of Arabidopsis PDF1.2 gene is regulated by jasmonic acid (JA) and ethylene (ET). It also has been well documented that GCC box is an element re- sponsive to ET,however, the responsive mechanism of JA in such plant defense gene expression is unclear. In this paper, the authors define the essential cis-acting element in PDF1.2 promoter responsive to methyl jasmonate (MeJA) through fragment deletions and site-directed mutageneses combining Agrobacterium-mediated transient reporter gene expression in tobacco leaves. Firstly, the MeJA inducible expression of PDF1.2 was confirmed by using the upstream ?1.86 kb fragment of PDF1.2 gene. Secondly, the upstream –300— ?243 bp fragment of the promoter was evidenced to respond to MeJA. To further characterize this promoter region, three point mutations were introduced into the –300—?243 bp fragment of the promoter. This result showed that the muta- tion of GCC box abolished MeJA induction, whereas the mutations of the G box-like and the imperfect palindrome sequence did not significantly decrease MeJA inducible effect, indicating that GCC box in PDF1.2 is essential for MeJA induction. The sufficient responsiveness to MeJA of this GCC box was further investigated by 4×GCC fused up- stream to the CaMV 35S minimal promoter. This result sug- gested that the fused promoter was able to activate reporter gene expression in response to MeJA. Thus these results in- dicate that the GCC box in PDF1.2 is an essential and suffi- cient element to confer MeJA induction.展开更多
文摘【目的】构建ERF(ethylene-responsive element binding factor)转录因子基因W17的亚细胞定位载体和原核表达载体,验证W17是否具有核定位功能,阐明W17与GCC、DRE探针的体外结合特性,利用GUS瞬时表达系统分析W17蛋白的体内结合特性和转录激活功能,初步预测W17在植物胁迫信号传导途径中的作用。【方法】构建W17/163hGFP亚细胞定位载体,基因枪转化洋葱表皮细胞,暗培养24h后共聚焦显微镜下观察。构建W17/pGEX-4T-1原核表达载体,转入大肠杆菌BL21(DE3),IPTG(0.5mmol·L-1,3h)诱导,GST纯化柱纯化,纯化的融合蛋白与[γ-32P]ATP标记的GCC、DRE探针混合进行凝胶阻滞试验。构建GUS瞬时表达系统,通过农杆菌介导转化烟草,X-Gluc染色、酒精脱色后体视显微镜下观察。【结果】W17基因具有核定位功能,纯化的融合蛋白GST/W17能与正常GCC、DRE探针体外特异结合,与突变GCC、DRE探针不结合,在植物体内与GCC特异结合并能激活下游GUS基因表达。【结论】W17通过自身的NLS进入核内行使功能,参与了GCC-box调控的生物胁迫信号传导途径,还可能参与了非生物胁迫(盐胁迫)传导途径。
基金financially supported by the National 863 Program of China(2007AA10Z130)the National Natural Science Foundation of China(30700504).
文摘The study aims to detect the subcellular localization of ERF （ethylene-responsive element binding factor） transcription factor W17 protein, the interaction between W 17 and cis-acting regulatory elements GCC-box and DRE in vitro, the binding and transactivating ability in vivo, and the role of W17 in higher plant stress-signal pathway. Recombinant plasmid W17/163hGFP was introduced into onion epidermal cells by the particle bombardment method with a PDS 1000/He. Transformed cells were incubated for 24 h at 22℃ in the dark and green fluorescence was monitored under a confocal microscope. The gene W17 was fused N-terminus of GST （glutathione-S-transferase） in prokaryotic expression vector pGEX-4T-1 and then transformed into E. coli strain BL21 （DE3）. IPTG （0.5 mmol L-1） was added to induce the expression of recombinant GST/W17 for 3 h. The fused proteins were purified by GST purification columns, and then subjected to gel retardation assay with a 32p-labeled GCC or DRE sequence. The different reporter and effector plasmids were introduced into tobacco leaves through agroinfiltration, then transformed leaves stained by X-Gluc, faded with 75% alcohol and monitored under a Stereozooming microscope. The GFP fused with W17 protein was localized in the nuclei; SDS-PAGE assay demonstrated that the fused protein GST/W17 could be induced and purified with molecular weight at around 42.2 kD under the induction of IPTG. Purified fused protein was able to specifically bind to both the wild-type GCC-box and DRE element, but had no interaction with either the mutant DRE or GCC-box; W17 protein can bind to GCC-box and transactive downstream GUS gene in vivo. W17 can localize into the nuclei, and it may be involved not only in biotic stresses controlled by GCC-box, but also in abiotic stresses （e.g., salt-） induced signaling pathway.
文摘The expression of Arabidopsis PDF1.2 gene is regulated by jasmonic acid (JA) and ethylene (ET). It also has been well documented that GCC box is an element re- sponsive to ET,however, the responsive mechanism of JA in such plant defense gene expression is unclear. In this paper, the authors define the essential cis-acting element in PDF1.2 promoter responsive to methyl jasmonate (MeJA) through fragment deletions and site-directed mutageneses combining Agrobacterium-mediated transient reporter gene expression in tobacco leaves. Firstly, the MeJA inducible expression of PDF1.2 was confirmed by using the upstream ?1.86 kb fragment of PDF1.2 gene. Secondly, the upstream –300— ?243 bp fragment of the promoter was evidenced to respond to MeJA. To further characterize this promoter region, three point mutations were introduced into the –300—?243 bp fragment of the promoter. This result showed that the muta- tion of GCC box abolished MeJA induction, whereas the mutations of the G box-like and the imperfect palindrome sequence did not significantly decrease MeJA inducible effect, indicating that GCC box in PDF1.2 is essential for MeJA induction. The sufficient responsiveness to MeJA of this GCC box was further investigated by 4×GCC fused up- stream to the CaMV 35S minimal promoter. This result sug- gested that the fused promoter was able to activate reporter gene expression in response to MeJA. Thus these results in- dicate that the GCC box in PDF1.2 is an essential and suffi- cient element to confer MeJA induction.
