單胃動物體內由於缺乏可分解植酸(phytate)的酵素，所以無法將穀物飼料中的植酸消化吸收。因此，畜牧業者廣泛使用微生物發酵所生產的植酸酶(phytase)作為飼料添加。植酸酶可以將植酸分解成肌醇(myo-inositol)及無機磷酸，提升動物對植物性磷酸鹽及其它營養素之攝取。然而，以發酵的方式來生產植酸酶會有成本過高的問題。
本實驗室過去的研究顯示溫泉菌（Thermus thermophilus）的酸性磷酸酶(acid phosphatase)具有水解植酸之能力(譚, 2009)。故本研究的目的為利用轉殖植物來生產磷酸酶。首先建構二個表現載體(pBIN-IV-AP1及pBIN-IV-AP2)，再利用農桿菌將表現載體導入菸草中，分別將蛋白質表現於胞內(pBIN-IV-AP1轉殖品系)或使其分泌至胞外(pBIN-IV-AP2轉殖品系)。利用PCR篩選轉殖植物(T0及T1)，顯示有外源基因存在，但是Southern blot分析T1轉殖植物的結果並不能清楚地偵測外源基因的copy數目。另外，用Western blot偵測蛋白質的表現量，在轉殖植物與對照組並未有顯著差異；zymogram分析結果也顯示某些轉殖植物雖有表現外源蛋白但其表現量低。此外，Northern blot的結果顯示RNA的表現量低。綜合本實驗結果，可推測轉殖植物確實帶有AP基因，但是因為基因表現量低及實驗技術問題因此轉殖植物與非轉殖植物的實驗結果沒有顯著差異。

Lack of phytate-hydrolyzing enzymes in monogastric animals’ digestive tracts and antinutritional effect of undigested phytate of animal feeds would limit the nutritional value of cereals. Phytase (myo-inosital hexakis-phosphate phosphohydrolase) can hydrolyze phytate to myo-inositol and inorganic phosphate. Therefore, phytase produced in microorganisms by fermentation have been widely used as a supplement to animal feed. However, the cost is relatively high for the production of phytase through fermentation.
From previous studies in our Lab., the putative acid phosphatase from Thermus thermophilus is able to hydrolyze phytate (Tham 2009). Therefore, the aim of this study is to highly produce acid phosphatase in transgenic plants. First, two plant nuclear expression vectors were constructed, pBIN-IV-AP1 and pBIN-IV-AP2, respectively. The former can direct the expressed acid phosphatase to cytosol and the latter can direct the expressed acid phosphatase to apoplast. Subsequently, these two expression vectors were individually transformed into tobacco by Agrobacterium–mediated method.
T0 and T1 transgenic plants were analyzed by PCR to confirm the integration of foreign gene into chromosome. However, Southern blot analysis of T1 transgenic plants did not clearly detect the copy number of foreign gene integration into chromosome. From the results of immuno-detection, it revealed that there were no significant differences between wild-type and transgenic plants in terms of acid phosphatase expression level. In contrast, zymogram assay indicated that acid phosphatase was expressed in some transgenic plants, but their expression levels are low. Furthermore, Northern blot results suggested that the RNA expression levels are low in selected transgenic plants. In conclusion, transgenic plants indeed carried AP gene in their genome, but their expression level is too low to be clearly distinguished from wild type.

譚思潔 (2009) Thermus thermophilus 的磷酸酶之生化特性研究. 國立成功大學生物科技研究所碩士論文

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