植物的生長發育除了需要大量的碳水化合物之外，氮素也是維持生理生化機能不可或缺的重要營養因子。植物除了自外界吸收氮源外，也靠蛋白(proteinase)分解體內的貯存性蛋白質(storage protein)或其它型式的蛋白質供給氮源(nitrogen source)，而cysteine proteinase (CysP)便是分解此類蛋白質最主要的酵素。
本實驗室的研究已發現，種子萌芽、老化、GA3及缺氮都會誘導水稻OsEP3A( Oryza sativa endoproteinase 3A)基因的表現；它所衍譯的蛋白質REP-1是一種cysteine proteinase（CysP），可分解水稻種子主要的儲藏性蛋白質glutelin，以提供種子發芽所需的大量氮源。而缺氮10天的水稻懸浮培養細胞，其OsEP3A mRNA也會大量的累積。顯示OsEP3A基因大約長1kb的啟動子(promoter)區域包含著受氮素調控的區域(nitrogen response sequence, NRS)。
為了更進一步了解OsEP3A promoter(啟動子)受到氮素調控的重要區域，我們將OsEP3A基因的5’端做系列漸次縮短(Serial deletion)，再利用農桿菌(Agrobacterium)感染植物的基因轉殖技術，將6個不同長度的OsEP3A啟動子/gusA的嵌合基因轉殖到水稻癒傷組織，並得到57個成功的轉殖細胞系。經由北方墨點法及GUS活性分析的結果，證明OsEP3A啟動子受到氮素所調控的位置(NRS)在轉錄起始位上游278~205 bp，並且可能包含負調控的區域(negative regulation domain)。此外，GUS染色法分析轉殖細胞系的結果，也顯示啟動子的活性的確受到氮素所調控，同時也間接證明了水稻細胞於缺氮的環境下，將會分泌大量的CysP到細胞外，以尋找可利用的氮源。

Nitrogen and carbohydrate metabolisms are essential for growth and development in plant。Nitrogen is an essential macro-element of mineral nutrient。 In addition to absorb nitrogen source from outer environment， catabolism of protein and nucleic acid supply molecules to adjust the size of intracellular nitrogen pool. Cysteine proteinase (CysP) is well known as a major enzyme involved in the storage protein degradation。
Our previous results show that a REP-1 proteinase in rice encoded by OsEP3A gene may play a role in various physiological responses and processes。OsEP3A gene expression is induced in the cultured rice cells by nitrogen starvation，and the accumulation of OsEP3A mRNA is suppressed by the addition of nitrogen source into the culture medium。 These results indicate that promoter region of the gene included about 1.0 kb upstream sequence might contain nitrogen response element (NRE)。
In this study，we are going to find out NRE in the OsEP3A gene promoter。 A OsEP3A promoter/gusA chimeric gene was constructed and a serial deletion of 5’-promoter sequence was performed。 Various length of OsEP3A promoter/gusA chimeric genes were introduced into rice cells by Agrobacterium transformation。 Fifty seven successful transformers were obtained by Hygromycin B selection。 According to the results of northern blot analysis and GUS activity determination，NRE should more likely localize in the region between upstream sequence –278 and –205 bp。 A derepression mode of OsEP3A gene expression is proposed in the response of nitrogen starvation。

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