Abstract
高等植物細胞的液泡包含二種主要的產電質子幫浦，分別是液泡質子腺核甘三磷酸水解酵素以及無機焦磷酸水解酵素，它們對於調節植物細胞的膨壓、維持細胞質恆定和代謝物的儲存具有相當的重要性。在此，我們針對綠豆液泡腺核甘三磷酸水解酵素A次体(V-ATPase A subunit) 基因作研究。為了選殖出這互補DNA (cDNA)，首先依照棉花及紅蘿蔔的 V-ATPase Asubunit 之具有保留性的序列，合成了引子(primer)，並利用從綠豆cDNA 庫所抽出的全部DNA 當作模版進行聚合酵素連鎖反應 (PCR)。而後再以PCR所得產物，當作探針到綠豆cDNA 庫中進行篩選。所選殖到的V-ATPase A subunit cDNA 分別與棉花、紅蘿蔔及油菜具有 83.8、83.4及81.1% 的相似性。這cDNA 序列可以轉譯成623個安基酸；推衍出的分子量為68,664 Da、等電點是5.17，且其安基酸序列和紅蘿蔔、棉花及大白菜有94.0~95.6% 的相同以及96.6~97.0% 的相似性。同時，我們也藉由北方轉漬雜合法(Northern hybridization)研究此A次体基因的表現。結果發現葉子表現量最多，其次是下胚軸，根部最基因組DNA 南方轉漬雜合法(Southern hybridization)分析，顯示此基因少。型態簡單，可能沒有異構基因(isoform) 的存在。為了更進一步的研究此A 次体的基因結構，我們於是著手構築綠豆基因庫。將基因組DNA 以酵素Sau3AI 進行部份截切，並以蔗糖梯度離心方式將DNA分成幾部份，其中取9 到20 kb 的DNA與l/DashII DNA進行結合並包裹。隨後並利用A次体的cDNA當作探針進行篩選。結果我們得到多個有訊號的殖株，其中有些殖株同時包含A次体基因的5 端和3 端區域。我們認為這些殖株應包含了整個A 次体的基因。Vacuoles of higher plant cells contain two primary electrogenicproton pumps, a vacuolar H+-ATPase (V-ATPase) and a H+-translocating inorganic pyrophosphatase (V-PPase), for theregulation of cell turgor, cytoplasmic homeostasis, and thestorage of metabolites. In this work, the structure of the mungbean V-ATPase A subunit gene was investigated. In order toclone the cDNA, primers were synthesized according to theconserved sequences of cotton and carrot V-ATPase A subunit toconduct polymerase chain reaction (PCR) using the total DNAextracted from mung bean cDNA library as a template. The PCRproduct was then employed as a probe to screen the constructedmung bean cDNA library. The cloned V-ATPase A subunit cDNAexhibits 83.8, 83.4, and 81.1% nucleotide homology to those ofcotton, carrot, and Brassica napus, respectively. The cDNAsequence encodes 623 amino acids with a predicted Mr of68,664and a predicted isoelectric point of 5.17. The amino acidsequence of V-ATPase A subunit from mung bean seedlings shares94.0~95.0% identity and 96.6~97.0% similarity to those fromcarrot, cotton, and Brassica napus. Expression of the A subunitgene was also investigated by Northern hybridization. It wasfound that the leave expressed the most abundance of thetranscript, followed by hypocotyl and roots. Genomic Southernanalyses reveal a simple reaction pattern of the gene,referring the lack of isoform for the A subunit. We also tookfurther step to explore the genomic structure of the A subunit.To construct the DNA library, mung bean genomic DNA waspartially digested by Sau3AI, and fractionated by sucrosegradient centrifugation. The 9 to 20 kb DNAs were ligated withl/Dash II DNA and packaged. The constructed library wasscreened using A subunit cDNA as a probe, and several positiveclones were obtained. Some of these clones contain both 5□and3□regions of the gene.