臺灣博碩士論文加值系統

在種子發育過程中，有一類蛋白質會大量累積於種子成熟後期，並於種子萌芽後消失，故被稱為 LEA (late embryogenesis abundant ) 蛋白質，此類蛋白質也稱為種子成熟蛋白，在單子葉及雙子葉植物中皆已廣泛地被發表。大豆種子成熟蛋白GmPM殖系已陸續被定序出41個選殖株，在本文中，將對GmPM9之啟動子活性及GmPM1、GmPM2及GmPM8之蛋白質功能加以探討，以轉殖阿拉伯芥為策略，研究大豆種子成熟蛋白基因之調控與表現作用。在調控研究方面，將GmPM9之啟動子區域切割成不等長度的五個片段後，把這些片段構築於GUS (β-glucuronidase)基因上游，成為農桿菌(Agrobacterium tumefaciens)之轉殖載體，再轉殖入阿拉伯芥中，結果顯示 GUS 活性僅被偵測於轉殖阿拉伯芥之成熟種子及之幼苗之子葉與下胚軸部份。種子發育期間，各啟動子片段皆於開花十天後開始累積 GUS 活性於種子中，並在開花十四天後種子成熟時達到最高值。利用螢光定量分析顯示最長之啟動子區域 (-966 bp 至 +91 bp) 可獲得最高之GUS 表現值，而最短之啟動子片段 (-114 bp 至 +91 bp) 足以使報導基因表現於種子中。對轉殖植株施以 PEG、mannitol 及 NaCl 處理，可誘導 GUS 活性表現於葉片，但外加 ABA、低溫及傷害處理則無法誘導其表現。上述結果顯示 GmPM9 之啟動子具有種子專一性，適合將其利用於研究種子專一性之課題上。在蛋白質功能研究部分，則以 CaMV 35S 啟動子分別驅動 GmPM 蛋白質表現於轉型阿拉伯芥中，所得之轉型植株可持續表現 GmPM1、GmPM2 或 GmPM8 蛋白質，分別稱為 TAZ2、TAZ16 及 TAZ238 。以轉型阿拉伯芥測試其耐逆境之能力，得知其生長狀況及外表型與未轉型種子相同。以雜交方式使轉型阿拉伯芥能同時表現二或三種 GmPM 蛋白質於同一植株中，其抗逆境之能力亦與未轉型植株相同。以免疫金粒定位 GmPM 蛋白質之結果顯示，GmPM1 蛋白質表現於轉型阿拉伯芥之細胞質及細胞核中，GmPM2 蛋白質存在於細胞質中，而GmPM8 蛋白質則出現於細胞質及澱粉粒中。保存於4 ℃之 TAZ 種子經過四年後仍保有約9成之發芽率，而未轉型之種子及 TAZP 系列(見第一章)之種子則幾乎全數死亡，種子之存活與 GmPM 蛋白質之表現是否有關，仍需進一步之確認。

Late embryogenesis abundant (LEA) proteins are synthesized during the late stages of seed development, and have been widely reported in monocot and dicot plants. In order to understand the developmental regulation and protein function of the soybean GmPM genes encoding LEA proteins, we used the transgenic Arabidopsis as tools in this study. For developmental regulation, a series of fragments of the promoter region were fused to the β-glucuronidase (GUS) reporter gene (uirA), to an Agrobacterium transgenic vector, and transformed the resulting constructs (pZP966, pZP572, pZP510, pZP294 and pZP114) into Arabidopsis thaliana. GUS enzyme activities were detected only in mature seeds, and cotyledons and hypocotyls of seedlings in transgenic Arabidopsis containing any one of the five constructs; they were not detected in other tissues at different developmental stages. During seed development, GUS activity detected at 10 days after flowering (DAF) and increased rapidly to a maximum in the mature seeds at 14 DAF. The longest promoter construct (pZP966) enabled the transgenic plants to exhibit the highest GUS activity, whereas the shortest construct (pZP114) was sufficient to direct the expression of the GUS gene at a detectable level. These findings indicate that the promoter of GmPM9 can be used to express desired genes in seeds during late seed maturation. The expression of the GUS gene could be induced in the leaves of the transgenic plants by salt and desiccation, but not by ABA, cold and wounding treatment. For protein function study, the GmPM1, GmPM2 and GmPM8 are seed maturation proteins of soybean, which were introduced into Arabidopsis by vacuum transformation method independently. Expression of these GmPM genes were regulated by CaMV 35S promoter and the proteins could constitutive accumulate in transgenic Arabidopsis. Cellular localizations of GmPM proteins were detected by immunogold. GmPM1 proteins were found in both cytoplasm and nucleus, GmPM2 proteins were accumulated in cytoplasm and GmPM8 proteins were in cytoplasm and starch grains. The transgenic Arabidopsis were subjected to osmotic and salt stress, there were no difference in the phenotype between transgenic and untransformed plants. These results suggested that GmPM proteins were not sufficient to increase stress tolerance in transgenic Arabidopsis. The untransformed seeds stored at 4℃ for four years loss the germination ability, but the seeds of TAZ2, TAZ16 and TAZ238 stored at the same condition could germinate at high percentage, however, the correlation between GmPM proteins and seed longevity needs to be further examined.

封面
中文摘要
英文摘要
前人研究
第一章 大豆種子成熟蛋白基因GmPM9在阿拉伯芥中調控之研究
壹、前言
貳、材料與方法
參、結果
肆、討論
伍、圖表
第二章 大豆種子成熟蛋白在轉殖阿拉伯芥中之表現
壹、前言
貳、材料與方法
參、結果
肆、討論
伍、圖表
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