Abstract
在植物細胞的液泡膜上同時存在著兩種氫質子傳遞酵素，分別是液泡腺核甘三磷酸水解酵素 (V-ATPase) 和液泡焦磷酸水解酵素(V-PPase)， 由這兩種酵素所產生的氫質子梯度是種很重要的驅動力量，可用來推動一連串的生理功能。現已知這兩種酵素具有等值的能力產生氫質子梯度，這引起我們極大的興趣去探索它們之間表現與其調控的差異。 在本實驗中，我們首先想從綠豆cDNA 庫中選殖出肌動蛋白 (actin) 當做內對照(internalcontrol)。 我們設計了兩個聚合酵素連鎖反應 (PCR) 的引子(primer) 從綠豆 cDNA 庫中擴增出 0.94-kb 肌動蛋白的片段。接著，以此片段當作探針再到綠豆 cD NA 庫中進行篩選。 將所選殖到的 actincDNA 部份定序後發現與豌豆、水稻及紅蘿蔔有80%、79% 及 76% 的相似性。在基因表現研究上，當將兩天大的綠豆種苗培養在不同濃度的磷酸鹽(Na2HPO4) 中，三天後， 選用下胚軸分離出液泡膜。我們發現隨著磷酸鹽濃度上升液泡焦磷酸水解酵素的活性有下降的現象，而液泡腺核甘三磷酸水解酵素則沒有明顯變化。 不過， 我們由西方轉漬法(Westernblotting) 卻發現這兩種酵素蛋白質的量並沒有明顯的變化。我們也進一步想從北方轉漬法 (Northern blotting) 觀察 mRNA是否也會連帶變化。 我們發現當選用 (NH4)2HPO4、 CaCl2 和Ca(N03)2 培育綠豆種苗時， 這兩種酵素在下胚質軸的 mRNA 量並不會顯著變化。 然而，當以 CaCl2 處理時，液泡腺核甘三磷酸水解酵素在根部的 mRNA 量卻有三倍的增加，液泡焦磷酸水解酵素則沒明顯變化。同時，我們也發現這兩種酵素在不同組織的mRNA 表現有顯著差異， 在下胚軸的表現量都最多，根部最少。另外，我們也初步探討其它環境因子的影響效應。當以熱處理時，液泡焦磷酸水解酵素的 mRNA 表現量呈現三倍的下降，而液泡腺核甘三磷酸水解酵素只有兩倍下降。至於這兩種酵素真正的調控機制為何，由現有證據還無法推測，我們可能還需要設計一連串實驗才能夠加以釐清。Plant cells contain two H(-pumping enzymes, V-ATPase andV-PPase, residing on the membrane of the vacuole. Theprotongradient produced by these enzymes are importantdrivingforce for maintaining cellular function. It is widelyknownthese two enzymes generate approximately equalprotongradients across the vacuolar membrane. In this study,weattempted to explore why there exists two parallel H( pumpsontonoplast of plant cells simultaneously. For calibration,wefirst cloned the cDNA sequence of actin f rom mung beanas theinternal control. Based on homology comparisonbetweenseveral species, we designed two PCR primers toamplify a0.94-kb fragment of actin from the mung bean cDNAlibrary. Thefragment was then employed as a probe to screenthe constructedmung bean cDNA library. Partial sequence ofcloned actin cDNAexhibits 80, 79, and 76% nucleotidehomology to those frompea, rice, and carrot, respectively.For exploring the regulatorymechanism, 2-day-old seedlingswere grown at differentconcentrations of Na2HPO4. Afterdays, the activity of V-PPaseon hypocotyl declined underphosphate salt treatment, whilethat of the V-ATPaseremained constant. However, Westernblotting showed noapparent change in the protein levelsof V-PPase andV-ATPase A, B subunits. Furthermore, themRNA of theseenzymes from hypocotyl remained constant whenseedlings weretreated with CaCl2 and Ca(NO3)2. Nevertheless,CaCl2 stimulated 3-fold increase in the transcript of V-ATPaseA subunitfrom the root. We found both V-ATPase and V-PPaseexhibitedspecific regulation in different tissues. The mRNAabundanceof V-ATPase A subunit was about 2-fold higher inhypocotylthan in leaf and root, and V-PPase also about 2-foldhigherin hypocotyl and leaf than in root. In theotherenvironmental study, under heat treatment therelativeamount of mRNA were decreased by 3 and 2 fold for V-PPaseand V-ATPase A subunit, respectively. In summary, westilldo not know exactly how the genes for bothtonoplastH(-pumping enzymes are regulated from our present work.