| Investigating Localization of Chimeric Transporter Proteins within Chloroplasts of Arabidopsis thaliana
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Author:
Date:
2018-02-05
[Abstract] In this protocol, we describe a method to design chimeric proteins for specific targeting to the inner envelope membrane (IEM) of Arabidopsis chloroplasts and the confirmation of their localization by biochemical analysis. Specific targeting to the chloroplast IEM can be achieved by fusing the protein of interest with a transit peptide and an IEM targeting signal. This protocol makes it possible to investigate the localization of chimeric proteins in chloroplasts using a small number of transgenic plants by using a modified method of chloroplast isolation and fractionation. IEM localization of chimeric proteins can be further assessed by trypsin digestion and alkaline extraction. Here, the localization of the chimeric bicarbonate transporter, designated as SbtAII, is detected by ...
[摘要] 在这个协议中，我们描述了一种设计嵌合蛋白的方法，用于特异性靶向拟南芥叶绿体的内包膜（IEM）并通过生化分析确定它们的定位。 叶绿体IEM的特异性靶向可通过将感兴趣的蛋白质与转运肽和IEM靶向信号融合来实现。 这个协议使得有可能使用少量的转基因植物，通过使用修改的叶绿体分离和分离方法来研究嵌合蛋白在叶绿体中的定位。 嵌合蛋白的IEM定位可以通过胰蛋白酶消化和碱性提取进一步评估。 在此，称为SbtAII的嵌合碳酸氢根转运蛋白的定位通过使用针对葡萄球菌蛋白A的抗体进行蛋白质印迹来检测。该方案改编自上原等人，2016年
【背景】有人提出将蓝藻CO 2浓度机制整合到叶绿体中是改善C 3+植物光合作用的有希望的方法。 根据理论估计，将BicA和SbtA整合到叶绿体IEM中可以提高光合CO 2固定率。 我们研究了核编码的蓝细菌碳酸氢盐转运蛋白BicA和SbtA与拟南芥叶绿体的IEM的整合。 因此，我们制定了一个协议，设计嵌合构造为特定目标的IEM和调查嵌合蛋白在叶绿体中的定位。
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| Purification of Flagellin from Acidovorax avenae and Analysis of Plant Immune Responses Induced by the Purified Flagellin
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Author:
Date:
2016-08-20
[Abstract] Plants sense potential pathogens by recognizing conserved pathogen-associated molecular patterns (PAMPs) that cause PAMP-triggered immunity (PTI) including the generation of reactive oxygen species, callose deposition, and expression of several PTI-related genes. Acidovorax avenae is a Gam-negative bacterium that causes a seedling disease characterized by the deposition of brown stripes on the sheaths of infected plants. We previously reported that flagellin isolated from the rice avirulent A. avenae N1141 strain induces PTI, while flagellin isolated from the rice virulent A. avenae K1 strain does not induce PTI. To examine the molecular mechanism of specific PTI induction by N1141 flagellin, highly purified flagellin from N1141 or K1 strains is required. Here, ...
[摘要] 植物通过识别引起PAMP触发的免疫（PTI）的保守的病原体相关分子模式（PAMP）来感测潜在的病原体，包括活性氧的产生，胼lose质沉积和几个PTI相关基因的表达。 酸性弧菌是一种革兰氏阴性细菌，其导致幼苗疾病，其特征在于在感染的植物的鞘上沉积棕色条纹。 我们以前报告了从无毒力的大米中分离的鞭毛蛋白。 avenae N1141菌株诱导PTI，而从水稻毒力分离的鞭毛蛋白。 avenae K1株不诱导PTI。 为了检查N1141鞭毛蛋白的特异性PTI诱导的分子机制，需要来自N1141或K1菌株的高度纯化的鞭毛蛋白。 在这里，我们描述了一种高质量的纯化方法。 avenae鞭毛蛋白，并用于PTI诱导研究。
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