A toolset of aequorin expression vectors for in planta studies of subcellular calcium concentrations in Arabidopsis thaliana.
A toolset of aequorin expression vectors for in planta studies of subcellular calcium concentrations in Arabidopsis thaliana.
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DOI：
10.1093/jxb/err406
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发表时间：
2012-02
影响因子：
6.9
通讯作者：
Vothknecht UC
中科院分区：
文献类型：
--
作者：
Mehlmer N;Parvin N;Hurst CH;Knight MR;Teige M;Vothknecht UC
钙长期以来被认为是植物中最重要的信号成分之一。许多非生物和生物刺激通过细胞钙浓度的时间和空间变化转化为细胞反应，钙敏感蛋白水母发光蛋白已被用作遗传编码的钙指示剂，用于测量植物中的钙。这项工作的目的是生成一套兼容的水母发光蛋白表达质粒，用于生成转基因植物系，以测量不同细胞亚区室中钙水平的变化。水母发光蛋白与不同的靶向肽或细胞器蛋白融合，作为将其定位到细胞质、细胞核、质膜和线粒体的手段。此外，构建体被设计为将水母发光蛋白定位于基质以及叶绿体包膜的内表面和外表面。质粒的模块化设置还允许轻松更换靶向序列以包括其他区室。还包括额外的 YFP 融合，以通过激光扫描共聚焦显微镜验证所有构建体的正确亚细胞定位。对于每个构建体，制备由 35S 或 UBI10 启动子驱动的基于 pBin19 的二元表达载体，用于农杆菌介导的转化。产生了稳定的拟南芥品系，并且对几个品系的初步测试证实了它们在体内测量钙信号的可行性。
Calcium has long been acknowledged as one of the most important signalling components in plants. Many abiotic and biotic stimuli are transduced into a cellular response by temporal and spatial changes in cellular calcium concentration and the calcium-sensitive protein aequorin has been exploited as a genetically encoded calcium indicator for the measurement of calcium in planta. The objective of this work was to generate a compatible set of aequorin expression plasmids for the generation of transgenic plant lines to measure changes in calcium levels in different cellular subcompartments. Aequorin was fused to different targeting peptides or organellar proteins as a means to localize it to the cytosol, the nucleus, the plasma membrane, and the mitochondria. Furthermore, constructs were designed to localize aequorin in the stroma as well as the inner and outer surface of the chloroplast envelope membranes. The modular set-up of the plasmids also allows the easy replacement of targeting sequences to include other compartments. An additional YFP-fusion was included to verify the correct subcellular localization of all constructs by laser scanning confocal microscopy. For each construct, pBin19-based binary expression vectors driven by the 35S or UBI10 promoter were made for Agrobacterium-mediated transformation. Stable Arabidopsis lines were generated and initial tests of several lines confirmed their feasibility to measure calcium signals in vivo.
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影响因子：
5.4
作者：
Bussemer, Johanna;Chigri, Fatima;Vothknecht, Ute C.
通讯作者：
Vothknecht, Ute C.
影响因子：
4.8
作者：
Aseeva, E;Ossenbühl, F;Vothknecht, UC
通讯作者：
Vothknecht, UC
影响因子：
7.4
作者：
Dammann, C;Ichida, A;Harper, JF
通讯作者：
Harper, JF
影响因子：
7.2
作者：
Clough, SJ;Bent, AF
通讯作者：
Bent, AF
影响因子：
11.6
作者：
Abdel-Ghany, SE;Müller-Moulé, P;Shikanai, T
通讯作者：
Shikanai, T