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標題: Cloning of The Genes Expressed During Early Stages of Rice (Oryza sativa L.) Embryogenesis
作者: 廖仁盟
Liao, Ren-Mong
關鍵字: 水稻 台農67號;Rice (Oryza sativa L. cv. Tainung 67;胚發育;差異表現法;基因選殖;TNG67);Embryogenesis;Differential display;Gene cloning
出版社: 分子生物學研究所
Organ differentiation in higher plants occurrs at postembryonic stage or during germination, but its blueprint is built during embryogenesis. In order to unravel the molecular mechanism and understand the pattern formation during embryogenesis in higher plants. In this thesis, differential display method was applied to screen early embryogenesis related gene fragments. After differential screening, the PCR products from embryo/endosperm and endosperm amplified by 48 primer combinations were resolved on polyacrylamide gel. Twenty two fragments that appeared only in embryo/endosperm were excised, PCR reamplified, and cloned into a dT vector. These clones were analyzed by reverse northern hybridization to identify which was related to early embryogenesis. However, no true fragment was found. Direct DNA sequencing and northern hybridization analysis showed two fragments, AP3A1-1 and AP9A1-2, were specifically expressed in early embryo and were used as probes to screen cDNA library.
Five clones, 5D7-1, 5D18-1, 5D28-2, 5D35-1 and 5D47-1were obtained from screening a rice 5DAP immature seed cDNA library with the AP3A1-1 probe. By BLAST analysis of NCBI, no identical nucleotide sequence was found. However, their amino acid sequences were identical to barley trypsin/amylase inhibitor, human CGI-141 protein, Arabidopsis growth regulator-like protein, putative serine protease-like protein in Arabidopsis, and rat 40S ribosomal protein S9, respectively. The sixth clone, 5D3W-1, obtained by screening cDNA library with AP9A1-2 probe showed highly identity (91 %) to a histone H2A F/Z family protein of Arabidopsis, and was postulated to be a new member of this protein family.
Detection of temporal gene expression during seed development and spatial expression in various rice tissues by northern blot analysis showed that the 5D7-1 clone is an endosperm-specific gene but has not specific expression pattern in the other tissues. The 5D18-1 clone is expressed through seed development except the mature endosperm, and its expression also detected in continuly growing tissues such as young leaf and shoot tip. Gene expression is only detected in embryo and tissues with cell dividing characteristics by using 5D28-2 cDNA fragment as a probe. The 5D35-1 clone is highly expressed not only in 5DAP endosperm through seed development but also in shoot tip. The expression of 5D47-1 gene is increased in embryo but decreased in endosperm during seed development. No gene expression was detected in mature leaf and coleoptile in which protein synthesis is quiescent. The 5D3W-1 gene is expressed at early embryogenesis stages and in fast dividing tissues such as shoot tip and callus. The Southern hybridizations showed that these six early embryogenesis related clone are presented as either single gene or low copy number in the rice genome.

高等植物的器官分化雖發生於發芽之後,但其生長藍圖卻於胚發育時期即已建立,為解開植物胚發育之分子機制及瞭解器官形成模式,本論文選殖與水稻胚發育早期相關的基因,期能朝解開單子葉胚發育之謎邁進。利用差異表現法(differential display)篩選胚發育早期相關基因片段,經篩檢48個引子組合,獲得22個僅出現於胚/胚乳之差異表現片段,經回收與PCR擴增,選殖於dT載體上,進行反向北方雜交分析,並未得到胚發育早期相關基因片段。經DNA定序及北方雜交分析獲得AP3A1-1與AP9A1-2二個與胚發育早期相關基因片段,並做為探針篩選cDNA基因庫。
以AP3A1-1基因片段為探針篩選水稻未熟種子(授粉後5天)之cDNA基因庫,得到5D 7-1、5D 18-1、5D 28-2、5D 35-1及5D 47-1等5個殖系,經過定序及NCBI網站比對分析,這些殖系之核酸序列均無法獲得較可靠與其匹配之相似基因,而其胺基酸序列則分別與大麥trypsin/amylase inhibitor、人類CGI-141蛋白、阿拉伯芥生長調節蛋白(growth regulator protein)、阿拉伯芥serine protease及老鼠40S ribosomal protein S9相似。而以AP9A1-2為探針篩選到的殖系5D 3W-1則與阿拉伯芥histone H2A F/Z family胺基酸序列具有高度一致性(identity),應為histone H2A F/Z family蛋白之一新成員。
將這些殖系分別進行北方雜交分析以檢測基因於胚發育時期與植株組織表現情形。5D 7-1殖系為種子發育過程中胚乳特異基因,不具組織特異性;5D 18-1殖系除成熟胚乳外,在胚與胚乳發育過程中皆有表現,同時幼葉、莖頂分生組織等細胞繼續生長的組織也有此基因表現;5D 28-2殖系在細胞快速生長及分裂的種子發育過程及組織則偵測到此基因表現;5D 35-1殖系雖在種子發育過程中皆有表現,但於授粉後5天胚乳較強,此外在莖頂分生組織也有明顯表現;5D 47-1殖系在種子發育過程中的胚表現量有上揚趨勢,胚乳則成下降趨勢,而在無新蛋白合成的組織如成熟葉及芽鞘則無基因表現;在胚發育早期及細胞分裂快速的組織可偵測到5D3W-1殖系的基因表現。南方雜交分析結果顯示,上述六個殖系在基因組DNA中的套數都是屬於一到三套的低套數。
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