秈稻耐旱性之轉錄體分析

Abstract

耐旱機制是多數量性狀遺傳，受到不同基因型和環境的影響故不易探討，因此作物耐旱機制的完整探討須建立於生理與遺傳的整合性研究才能加速耐旱水稻的育成，而次世代定序的優點在於提供快速且全面性的轉錄體及代謝途徑概況，可初步提供資訊以進一步吻合前述的要求。 本研究利用5種對乾旱敏感程度不一的秈稻品系為材料，分別是SM13、SM47 (兩者為疊氮化鈉誘變IR64所產生的耐旱突變系) 以及IR64 (乾旱敏感)，再配合HY15及TCN1做為耐旱與敏感品種的對照。以23.3% PEG 6000模擬滲透壓-0.6 Mpa的乾旱逆境，處理3、24小時以及處理24小時後回復24小時，萃取地上部RNA後利用Illumina平台進行RNA-Seq，將基因表現量數據化後利用統計軟體篩選乾旱下顯著差異表現基因（differentially expressed genes, DEGs），接著利用基因本體（gene ontology）找出對乾旱顯著反應的基因功能。 結果顯示DEGs的功能會隨著乾旱處理時期以及基因型而改變，不同秈稻在乾旱初期的基因表現相似但24 hr及回復期則漸漸採取不同機制，特別是細胞溝通與細胞壁內的生理反應。而基因型間的差異以SM47較為明顯，SM47獨特的耐旱相關基因功能包含訊息傳遞及碳水化合物代謝路徑等，另外為探討IR64與SM47在本質上的差異，嘗試以生資軟體分析兩者地上部轉錄體間的SNP。 SM47的生長勢及耐旱生理指標均顯示其具有耐旱特性，推測可能是由於滲透調節物質以及活化氧族清除者的生合成。這些存在不同基因型間的DEGs均有可能為耐旱候選基因，然而仍需要進一步的研究以驗證其功能。這些結果期望能提供乾旱反應的背景知識並加速耐旱水稻的育成。

Background: Drought tolerance mechanism is a complex trait that involves multiple genes and affected by genotypes and environments. Because improved methods of cultivation could not enhance yield efficiently, it is faster to conduct molecular breeding for drought tolerant rice to maintain a constant food supply. This research aims at building up background knowledge for drought tolerance mechanism in indica rice by transcriptome analysis. Methods: The materials contained 5 types of indica rice which had different sensitivity to drought, including SM13 (highly drought tolerant), SM47 (moderate drought tolerant), IR64 (drought sensitive), HY15 (drought tolerant) and TCN1 (drought sensitive). SM13 and SM47 were sodium azide (NaN3)-induced IR64 mutant lines. We conducted -0.6 Mpa drought treatments by PEG 6000 for 3 hr, 24 hr and recovery 24 hr (normal hydroponic solution after drought treatments for 24hr) when the materials reached three-leaf stage. Digitized gene expressions were calculated and differentially expressed genes (DEGs) were identified by CLC bio and DESeq, respectively. Results: Gene ontology analysis suggested that some functions of DEGs were different among drought treatments and genotypes. The drought responses may be similar between 5 indica rice genotypes under early stage (3 hr) but different under 24hr and recovery. Drought related genes specific to SM47 involved in signaling transduction, carbohydrate metabolic process and other metabolic pathways. In addition, bioinformatics tool unravelled that SNP induced by NaN3 existed in some SM47 transcripts. Conclusions: SM47 seemed to be more drought tolerant than the others and had good growth potential. The ability of drought tolerance might be related to osmolytes and ROS scavenger biosynthesis. The different DEGs between materials which had different sensitivity to drought were suggested to the candidate genes for drought tolerance. However, further research is needed to confirm their functions. The results could provide a genetic reference to accelerate the selection and breeding of drought tolerant rice.