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

植物囊泡转运工程-选择性囊泡转运的分子机制及其在植物重组蛋白生产中的应用

基本信息

批准号：
14350436
负责人：
YOSHIDA Kazuya
金额：
$ 9.02万
依托单位：
Nara Institute of Science and Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

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.

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，在植物中参与多种次生代谢反应。特别是，辣根（Armoracia 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蛋白被证明是一个具有n端（NTPP）和c端前肽（CTPP）的前蛋白。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去除。利用转基因培养烟草细胞（Nicotiana tabacum, BY2）分析系统，研究了HRP C1a、NTPP和CTPP在确定水泡运输途径中的功能作用。在本研究中，我们发现NTPP和CTPP对于成熟的HRP C1a蛋白准确定位到囊泡运输系统的液泡是必要和充分的。在没有编码CTPP的DNA区域的情况下，表达HRP C1a基因的细胞可以分泌HRP C1a。我们还研究了HRP C1a的NTPP和CTPP在烟草植物中的作用。在用NTPP和CTPP表达HRP C1a基因的烟草植株中，HRP C1a在液泡中积累。当缺乏CTPP的HRP C1a表达时，活性酶分泌到外质体空间。并且，我们可以利用翻译增强子NtADH 5′-UTR增强HRP C1a的表达。此外，我们在BY2细胞中确定了重组C1a蛋白的聚糖结构为Man3FucXylGlcNAc，与内源性液泡糖蛋白结构相同。