Gene expression of aleurone beta-amylase in rice

澱粉是水稻穀粒中含量最多的儲存性養分，種子萌芽後可經由水解酵素分解澱粉的作用，而提供小苗初期生長所需的碳源和能源。在水稻中，已知β-澱粉酶是於種子萌發後de novo合成，至於表現的組織及其生理功能都還不是很清楚，所以，期望藉由了解水稻β-澱粉酶(L10346)的基因表現的組織及發育時期，來推測此遍在型的β-澱粉酶可能扮演的生理功能。本論文先以薄層層析法(TLC)的方法，確定在水稻葉子的澱粉酶酵素圖譜中存在的四個條帶，都是β-澱粉酶。再將35S::L10346以農桿菌為媒介感染TNG67的癒傷組織，以電泳分析，確定L10346的蛋白質產物的確具β-澱粉酶的活性，且為澱粉酶圖譜上之BA4。將L10346與不同物種的β-澱粉酶的蛋白質氨基酸序列進行比對分析，得知L10346解碼蛋白質與其他作物(Alfalfa、Wheat、Cowpea、Soybean、AtBYM1、Sweet potato)的β-澱粉酶在蛋白質氨基酸序列上都具有很高的相似度(約70%以上)，而與葉綠體型的β-澱粉酶相較則相似度較低。將TNG67之L10346啟動子接上GUS報導基因，利用農桿菌感染法轉殖於入TNG67中，分析水稻轉殖株，於種子萌芽時，在糊粉層、內子葉、地上部的小苗都可以看到GUS活性的表現；而在葉子中GUS活性的表現主要是在維管束組織的部位；種子發育時期，在母體組織中也可看到GUS活性的表現。顯示水稻β-澱粉酶基因(L10346)的啟動子在組織和發育上具有特殊的專一性。另外在了解了水稻澱粉酶活性圖譜中之BA4即是L10346後，藉由蛋白質電泳分析的方法得知L10346活性與葉片組織的年齡有密切關係。本論文亦分析在不同逆境或是荷爾蒙處理之下，水稻澱粉酶活性的差異。During the post-germination period, degradation of storage starch in cereals is critical to the viability of seedlings. Cereal β-amylases are important enzymes responsible for the mobilization of starch in germinating grains. In rice, β-amylase is synthesized de novo upon seed germination and is almost absent in dry seeds. However, detailed analyses on β-amylase expression in other tissues have not yet been performed. L10346 is a gene located in chromosome 7 which encodes the major β-amylase in germinating seeds. Analysis of the L10346 peptide sequence indicated that it is highly similar to other β-amylase sequences, including rye, maize, barley, arabidopsis, soybean, wheat, alfalfa, white, cowpea, sweet-potato (in the range of 72~88%). β-amylase activity was detected in the aleurone layers and scutella of 3-day germinating seeds. Polymorphism in rice amylases at early stage of seed germination was analyzed by zymogram. The temporal and spatial regulation of L10346 gene expression are presented by histochemical analysis of β-glucuronidase (GUS) activity in transgenic rice carrying L10346::gus fusion gene. GUS activity was detected in developing and germinating kernel. Subcellular localization study was performed by a fusion protein of β-amylase (L10346) and YFP. Fluorescence of YFP was present in both the cytosol and nucleus. Four major amylolytic enzymes were present in rice leaves and identified as β-amylase, namely BA1~4, based on the analysis of digestion products. The product of L10346 was assigned as the BA4. The decrease in BA4 activity was accompanied with the age gradient in rice leaves.中文摘要 ……………………………………………………… i 英文摘要 ……………………………………………………… ii 前言 ……………………………………………………… 1 材料與方法 ………………………………………… 7 結果 ……………………………………………………… 18 一、 水稻栽培種台農67號(TNG67)之β-澱粉酶 …… 18 二、 L10346的特性分析析 ..................... 20 三、 荷爾蒙、糖類、逆境及老化對水稻澱粉酶L10346活 性的影響 …………………………………………… 22 四、 水稻栽培種台農67號(TNG67)與日本晴(NPB)β-澱粉 酶基因之比較 ……………………………………… 24 討論 ………………………………………………………… 25 參考文獻 ……………………………………………………… 32 圖表 ……………………………………………………… 40 附錄 …………………………………………………………….. 5