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、植物和动物中，并且在植物中涉及多种次级代谢反应。特别地，辣根（Armoracia rusticana）根代表商业PRX生产的主要来源。prxC 1a基因编码辣根PRX（HRP）C，主要在辣根植物的根和茎中表达。HRP C1 a蛋白被证明是作为前蛋白合成的，其具有N-末端（NTPP）和C-末端前肽（CTPP）。这些可能负责细胞内定位或分泌的前肽在成熟蛋白产生之前或同时被除去。利用转基因烟草（Nicotiana tabacum，BY2）细胞分析系统，研究了HRP C1 a NTPP和CTPP在确定囊泡运输途径中的功能作用，发现NTPP和CTPP是准确定位成熟HRP C1 a蛋白到囊泡运输系统液泡的必要和充分条件。在不含CTPP编码区的细胞中表达HRP C1 a基因，也能分泌HRP C1 a。本文还研究了HRP C1 a的NTPP和CTPP在烟草中的作用。在转NTPP和CTPP基因烟草中，HRP C1 a在液泡中积累。当缺乏CTPP的HRP C1 a表达时，活性酶分泌到质外体空间。同时，我们还可以利用翻译增强子NtADH 5 '-UTR增强HRP C1 a的表达。进一步测定了重组C1 a蛋白在BY2细胞中的糖链结构为Man 3FucXylGlcNAc，与内源性液泡糖蛋白的糖链结构相同。

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.