Plant vesicular transport engineering-molecular mechanisms of selective vesicular transport and application for recombinant protein production in plants

过氧化物酶(Prx，EC 1.11.1.7)广泛分布于微生物、植物和动物体内，参与多种次生代谢反应。特别是，辣根(Aroracia Rusticana)是商业PRX生产的主要来源。编码辣根Prx(HRP)C的prxC1a基因主要在辣根植物的根和茎中表达。HRP C1a蛋白是由N端(NTPP)和C端前肽(CTPP)共同合成的前体蛋白。这些可能负责细胞内定位或分泌的前肽在成熟蛋白质产生之前或伴随着产生成熟蛋白质而被移除。本研究利用转基因烟草细胞分析系统研究了HRP C1ANTPP和CTPP在确定囊泡运输途径中的作用，发现NTPP和CTPP是将成熟的HRP C1a蛋白准确定位到囊泡运输系统液泡中的必要条件和充分条件。表达HRP C1a基因的细胞在没有编码CTPP的DNA区域的情况下可以分泌HrPC1a。我们还研究了HRP C1a的NTPP和CTPP在烟草中的作用。在转NTPP和CTPP的转基因烟草植株中，HRP C1a在液泡中积累。当缺乏CTPP的HRP C1a被表达时，活性酶被分泌到质外体间隙。并且，我们可以利用翻译增强子NtADH 5‘-UTR来增强HRP C1a的表达。此外，我们还确定了重组C1a蛋白在BY-2细胞中的糖链结构为Man3FucxylGlcNAc，其结构与内源空泡乙二醇蛋白的结构相同。

Peroxidases (PRX, EC 1.11.1.7) are widely distributed across microorganisms, plants, and animals, and in plants have been implicated in a variety of secondary metabolic reactions. In particular, horseradish (Armoracia rusticana) root represents the main source of commercial PRX production. The prxC1a gene, which encodes horseradish PRX (HRP) C, is expressed mainly in the roots and stems of the horseradish plant. HRP C1a protein was shown to be synthesized as a preprotein wish both an N-terminal (NTPP) and a C-terminal propeptide (CTPP). These propeptides, which might be responsible for intracellular localization or secretion, are removed before or concomitant with production of the mature protein. We investigated the functional role of HRP C1a NTPP and CTPP in the determination of the vesicular transport route, with using analytic system of transgenic cultured tobacco cells (Nicotiana tabacum, BY2).In this research, we found out that NTPP and CTPP are necessary and sufficient for accurate localization of mature HRP C1a protein to vacuoles of the vesicular transport system. HRP C1a could be secreted from cells expressing HRP C1a gene without the DNA region encoding the CTPP. We also investigated the roles of the NTPP and the CTPP of HRP C1a in the tobacco plant. In transgenic tobacco plants expressing the HRP C1a gene with NTPP and CTPP,HRP C1a was accumulated in the vacuole. When HRP C1a lacking the CTPP was expressed, active enzyme was secreted into the apoplast space. And, we could enhance the expression of HRP C1a by the use of translational enhancer, NtADH 5'-UTR. Furthermore, we determined the glycan structure of recombinant C1a protein in BY2 cells was Man3FucXylGlcNAc which was same structure as that of endogenous vacuolar glycol-proteins.