絲瓜果實韌皮部蛋白質的純化、鑑定與定位

Abstract

植物韌皮部存在著一群組織專一性的蛋白質，稱之為P-蛋白質。以光學顯微鏡觀察絲瓜（Luffa cylindrica）果實表層構造，可以發現維管束組織成網狀分佈於中果皮中。蛋白質厚片染色結果顯示在維管束的篩管細胞及外果皮細胞中有大量蛋白質。觀察電子顯微鏡下韌皮部的超微構造，有絲狀的蛋白質密佈在韌皮部的細胞中，而在其他細胞並無此類蛋白質。 瓜科植物具有篩管腔大、篩孔大及韌皮部溢流液中富含蛋白質等特性，常被用來當作分析韌皮部蛋白質的材料。收集絲瓜果實韌皮部溢流液，檢測出蛋白質含量平均每毫升溢流液中有69毫克。此溢流液在空氣中會聚合凝固成膠狀，必須加入抗氧化劑來避免凝集。在這韌皮部溢流液中同時具有多種酵素活性，以活性染法測知有過氧化酵素、酸性磷酸酵素及超氧歧化酵素的活性，其中以超氧歧化同功酵素種類最多。 SDS電泳分析韌皮部溢流液可以依分子量大小區分出四群主要蛋白質：第一群在97.6 kDa左右；第二群介於31 kDa到45 kDa間，有三個主要蛋白質；第三群介於21.5 kDa到31 kDa間，有兩個主要蛋白質；第四群則是小於21.5 kDa。選擇含量最多的24 kDa蛋白質進一步純化，並製備抗血清，此抗血清效價達十萬。經由免疫轉印法，得知絲瓜果實中的24 kDa蛋白質同時也存在於莖及葉部，且偵測到其native form蛋白質，進一步用免疫螢光及免疫電顯，定位出24 kDa蛋白質專一分佈在維管束組織的篩管細胞中，由此可知，24 kDa蛋白質為一物種專一及組織專一性的蛋白質。

P-proteins are groups of proteins which express specifically in the phloem. In the fine structure observation, Luffa cylindrica fruit has reticular vascular bundles which surround the outer layer of the fruit. Lots of proteins can be detected in the sieve element and parenchyma cell by Coomassie blue stain. The sieve element is characterized by the sieve plates and sieve pores. Filamentous proteins can be observed in the cytosol of sieve element. The fruit exudate of Luffa cylindrica contains a lots of proteins around 69 mg per ml. The exudate exposed in air coaggregates easily and it can be avoided by the addition of antioxidant reagents in preparation. In SDS discontinuous polyacrylamide gel electrophoresis, these proteins are separated into four groups according to their molecular weight: the first group contain a major protein band about 97.6 kDa; the secondary group are in three major bands between 31 kDa and 45 kDa; the third group have two major bands from 21.5 kDa to 31 kDa; and the last group are proteins which molecular weights are smaller than 21.5 kDa. Isozymes of peroxidase, acid phosphatase and superoxide dismutase are also found in the phloem exudate. The most abundant protein band, which is about 24.2 percents of total proteins with molecular weight of 24 kDa, is selected for the antiserum preparation. The titer of the antiserum is about one hundred thousand. By using the antiserum for western blotting, a protein band in native gel is detected, indicating that the 24 kDa protein is the component of this native form protein. To survey the existance of the 24 kDa protein in differet organ and species, this protein can be detected only in the Luffa cylindrica. From the observation of immunofluorescence and immuno-gold labeling, the 24 kDa protein is specific to vascular bundles and immunolocalized in the cytosol of sieve elements which contain a lots of filamentous proteins.