Yasuda Hiroshi, Hayashi Yuji, Jomori Takahito, Takaiwa Fumio
Department of Plant Biotechnology, National institute of Agrobiological Sciences, Tsukuba, Ibaraki, 305-8602 Japan.
Plant Cell Physiol. 2006 Jun;47(6):756-63. doi: 10.1093/pcp/pcj049. Epub 2006 Apr 13.
Glucagon-like peptide 1 (GLP-1) is a 30 amino acid peptide hormone involved in insulin stimulation that is dependent upon blood glucose levels. We have previously reported that when this short peptide gene was directly expressed under the control of a glutelin promoter and its signal peptide, it was not accumulated in transgenic rice seed due to gene silencing. However, when the modified GLP-1 (mGLP-1) gene was enlarged to 5xmGLP-1 (mGLPx5) by tandem repeat, no silencing was observed. The mGLPx5 peptide could be accumulated in rice seed and its localization was mainly limited to the endoplasmic reticulum (ER). We also investigated alternative cellular localization sites that would increase accumulation. The relationship between the expression level and localization was examined by attaching the chitinase signal peptide to mGLPx5 to direct it into the intercellular space (apoplast), or by expression as a fusion protein with glutelin by insertion into a variable region of the acidic subunit, thus directing the peptide to protein body II (PB II). Attachment of the KDEL ER retention signal to the 6xmGLP-1 (mGLPx6) or its fusion to the C-terminus of the 13 kDa prolamin directed the peptide to the ER or PB I, respectively. Unexpectedly, these results indicated that mGLPx5 without any signal except for the glutelin signal peptide was accumulated to the greatest extent in rice endosperm. It can thus be concluded that the ER is a suitable intracellular organelle for accumulation of mGLPx5 peptide.
胰高血糖素样肽1（GLP-1）是一种由30个氨基酸组成的肽激素，参与依赖血糖水平的胰岛素刺激过程。我们之前报道过，当这个短肽基因在谷蛋白启动子及其信号肽的控制下直接表达时，由于基因沉默，它并未在转基因水稻种子中积累。然而，当修饰后的GLP-1（mGLP-1）基因通过串联重复扩大为5xmGLP-1（mGLPx5）时，未观察到沉默现象。mGLPx5肽能够在水稻种子中积累，其定位主要局限于内质网（ER）。我们还研究了其他可能增加积累的细胞定位位点。通过将几丁质酶信号肽连接到mGLPx5上以将其导向细胞间隙（质外体），或者通过插入酸性亚基的可变区域与谷蛋白作为融合蛋白表达，从而将该肽导向蛋白体II（PB II），来检测表达水平与定位之间的关系。将KDEL内质网滞留信号连接到6xmGLP-1（mGLPx6）上或将其融合到13 kDa醇溶蛋白的C末端，分别将该肽导向内质网或蛋白体I（PB I）。出乎意料的是，这些结果表明，除了谷蛋白信号肽外没有任何信号的mGLPx5在水稻胚乳中积累程度最大。因此可以得出结论，内质网是积累mGLPx5肽的合适细胞内细胞器。
