以DNA微陣列分析slpslp突變水稻之幼花序基因表現

Abstract

水稻穎花發育會影響榖粒大小，進而影響最終產量。本研究嘗試尋找與內外穎發育有關的基因，試驗材料得自農業試驗所水稻研究室，係以疊氮化鈉(sodium azide)誘導之水稻穎花突變體。其突變性狀有兩種變化，一種是內外穎退化（slp, stunted lemma and palea），另一種是小穎花，內外穎只有正常穎花的一半。小穎花突變體自交後代會產生正常型穎花(SLPSLP)、小穎花突變體(SLPslp)及內外穎退化突變體(slpslp)，分離比約為1:2:1，符合孟德爾單一基因分離模式。本試驗分別剝取正常型穎花及內外穎退化突變型穎花水稻之幼花序(<5 mm)，萃取total RNA，利用高通量(high-throughput)的DNA微陣列分析技術，全面分析兩種型態幼花序基因表現之不同。 本試驗之微陣列分析，將正常型穎花幼花序(SLPSLP)設定為對照組，內外穎退化突變型穎花（slpslp）幼花序設定為實驗組，進行基因差異表現比較分析，獲得實驗組比對照組，表現上升兩倍以上的基因有322個，表現下降兩倍的基因有92個，分別各選取3個基因利用Q-PCR(quantitative PCR)，驗證微陣列試驗結果之正確性。本研究結果與劉(2003)使用cDNA-AFLP所篩選的差異表現基因，相互比較，發現其中僅有一個salT基因表現趨勢一致，都在slpslp幼花序會大量表現。進一步以基因功能性將差異表現之基因進行分群，上升表現之基因可分為18群，下降表現之基因可分為9群，其中各有一群為轉錄因子。有17個轉錄因子在slpslp幼花序有上升表現兩倍以上之情形；5個轉錄因子則被抑制表現兩倍以上，本研究進一步探討這些轉錄因子與slpslp幼花序異常表現基因之間的關係。此外，篩選slpslp幼花序的差異表現基因中，獲得一個MADS相關基因表現受到抑制及一個AUX/IAA相關基因會大量表現，推測可能與slpslp穎花發生異常有關。本研究將差異表現基因繪製成基因圖譜，觀察基因於染色體上之分佈是否有特別密集的區域，可能與突變基因有關，此結果可以幫助預測突變基因所在位置，提供後續試驗一個參考的方向。

The size and shape of lemma and palea can determine the rice grain size, and influence final yield of rice. In this study, a mutated rice induced by sodium azide, is used to study genes that regulate development of lemma and palea. This mutant has two different mutated phenotypes. The homozygous line shows stunned lemma and palea(slpslp), and heterozygous line shows small lemma and palea(SLPslp), which are only half in length compared to the normal one. Self-crossing of heterozygous line(SLPslp) can produce three different types of lemma/palea phenotype； one is normal(SLPSLP), another is SLPslp, and the other is slpslp. The segregation ratio is close to 1:2:1, which fits to single gene model according to the Mendel’s first law. To investigate the difference of gene expression between SLPSLP and slpslp, young inflorescences(< 5 mm) were collected and used for DNA microarray analysis. From DNA microarray result, 322 up-regulated genes and 92 down-regulated genes were obtained by screening the gene expression with over 2-fold chang between slpslp and SLPSLP inflorescences. The microarray result was confirmed by using Q-PCR of 6 genes in up-or down-regulated gene categories. Comparing both results from DNA microarray and cDNA-AFLP(Liu, 2003), it was found that only salT gene was consistently highly expressed in slpslp inflorescence. After annotation, there are 126 and 40 known genes up-regulated and down-regulated respectively. Furthermore, 126 up-regulated gene were classified into 18 groups, 40 down-regulated gene were classified into 9 groups. In this study, we focused on transcription factors(TF) and analysed their relationship among differentially expressed genes. As so far, it needs more evidence to proof this relationship. However, there are two genes, OsMADS55 and OsIAA9, inhibited and enhanced, respectively in slpslp inflorescence might have effects on lemma/palea development and are worth studying further. Therefore, we made a gene map that descripe the differential expression gene distributing on chromosomes. This map can help to realize the location on chromosome of the mutant gene.