利用差異表現法選殖水稻H2A基因

Abstract

高等植物的器官分化雖發生於發芽之後，但其生長藍圖卻早於胚發育時期即已建立，為瞭解植物胚發育之分子機制及器官形成模式與基因表現，本文利用差異表現法 (differential display) 選殖與水稻胚發育早期相關的基因，以探討單子葉植物胚發育之謎津。利用差異表現法篩選胚發育早期相關基因片段，共計篩檢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，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，此或許是因反向北方雜交之效率所致，未能偵測到表現量較少，包括調節性之基囚等。經DNA定序及北方雜交分析獲得與胚發育早期相關基因片段AP9A2，並以之做為探針進行早胚之cDNA基因庫篩選所篩選到的殖系5D 3W-1(Accession No. AF193345)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。分析其在胚發育時期與各組織之基因表現，發現嘆在胚發育早期及細胞分裂快速的組織，可偵測到H2A基因的表現南方雜交分析結果顯示，此一基因在水稻基因組DNA中屬單套基因。 In higher plants, organ differentiation occurrs at postembryonic stage or during germination, however, its blueprint is built up during embryogenesis. In this report, differential display method (DDRT-PCR) was applied to screen early embryogenesis related gene fragments in order to unravel the molecular mechanism and understand the pattern formation during embryogenesis in rice. 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. No cDNA fragment was found to be related to early embryogenesis by reverse northem analysis. This may due to the poor labeling of cDNA probes by reverse transcription. Results of DNA sequencing and northern hybridization showed that fragment, AP9A1-2, was specifically expressed in the early embryo and used as a probe to screen a immature rice seed cDNA library. A clone, 5D3W-l, obtained by cDNA library screening 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. The 5D3W-l (Accession No. AF193345) gene is expressed at the early embryogenesis stages and in fast dividing tissues such as shoot tips and callus. The Southern hybridizations showed that this embryogenesis related clone is presented as a single gene in the rice genome.