Cloning of a tobacco apoplasmic invertase inhibitor - Proof of function of the recombinant protein and expression analysis during plant development
Cloning of a tobacco apoplasmic invertase inhibitor - Proof of function of the recombinant protein and expression analysis during plant development
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DOI：
10.1104/pp.116.2.733
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发表时间：
1998-02-01
期刊：
影响因子：
7.4
通讯作者：
Rausch, T
中科院分区：
文献类型：
--
作者：
Greiner, S;Krausgrill, S;Rausch, T
高等植物表达液泡和细胞壁转化酶（CWI）的几种亚型，其中一些在植物发育的某些阶段被抑制蛋白灭活。我们对烟草CWI的质外质抑制剂（INH）进行了纯化。基于胰蛋白酶片段的序列，我们通过逆转录-聚合酶链反应分离了全长INH编码cDNA克隆（Nt-inh 1）。Southern-blot分析显示INH由单拷贝或低拷贝基因编码。与拟南芥和温州蜜柑的表达序列标签克隆的比较表明，在其他植物中存在Nt-inh 1相关蛋白。重组Nt-inh 1编码的蛋白抑制烟草和红藜悬浮培养细胞的CWI和番茄果实的液泡转化酶，而酵母转化酶不受影响。然而，只有在同源系统中，抑制作用受Suc浓度的调节，如先前对从烟草细胞分离的INH所示。INH与CWI的高度特异性结合可通过固定化重组Nt-inh 1蛋白上的总细胞壁蛋白级分的亲和层析来显示。对Nt-inh 1和CWI在烟草植株不同部位的相对转录比例进行RNA-blot分析，结果表明，Nt-inh 1和CWI的表达并不总是协调的。
Higher plants express several isoforms of vacuolar and cell wall invertases (CWI), some of which are inactivated by inhibitory proteins at certain stages of plant development. We have purified an apoplasmic inhibitor (INH) of tobacco (Nicotiana tabacum) CWI to homogeneity. Based on sequences from tryptic fragments, we have isolated a full-length INH-encoding cDNA clone (Nt-inh1) via a reverse transcriptase-polymerase chain reaction. Southern-blot analysis revealed that INH is encoded by a single-or low-copy gene. Comparison with expressed sequence tag clones from Arabidopsis thaliana and Citrus unshiu indicated the presence of Nt-inh1-related proteins in other plants. The recombinant Nt-inh1-encoded protein inhibits CWI from tobacco and Chenopodium rubrum suspension-cultured cells and vacuolar invertase from tomato (Lycopersicon esculentum) fruit, whereas yeast invertase is not affected. However, only in the homologous system is the inhibition modulated by the concentration of Suc as previously shown for INH isolated from tobacco cells. Highly specific binding of INH to CWI could be shown by affinity chromatography of a total cell wall protein fraction on immobilized recombinant Nt-inh1 protein. RNA-blot analysis of relative transcript ratios for Nt-inh1 and CWI in different parts of adult tobacco plants revealed that the expression of both proteins is not always coordinate.