從三種水稻品系轉錄體分析探討抗淹水逆境之調控機制

Genome-wide transcriptomic profiles reveal comprehensive insights into flooding-tolerant responses in 3 rice cultivars

楊弘安(Hung-An Yang)

指導教授 ： 施明哲

國立臺灣大學/生命科學院/植物科學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201610114

摘要

SUB1A-1為第七群乙烯反應轉錄因子(ethylene response transcription factor, ERF)的成員之一，其基因表現會在淹水時被大量誘導，在水稻的抗淹水逆境中扮演了重要角色。根據過去的文獻指出，帶有SUB1A-1的水稻品系可以忍受長時間的淹水逆境。FR13A與Pokkali皆帶有SUB1A-1基因，因而能耐受淹水逆境，IR29帶有SUB1A-2，並無法耐受淹水。然而，都帶有SUB1A-1基因的FR13A與Pokkali在淹水時卻有截然不同的表現型，FR13A在淹水時，SUB1A-1的表現量會被大量誘導，使其停止生長以利節省能量。Pokkali的SUB1A-1的表現量在淹水時僅被些微誘導，植株會繼續生長，有趣的是Pokkali中低量表現的SUB1A-1仍具有功效，受到SUB1A-1所調控的醣解作用相關基因在Pokkali中也有被大量誘導表現，更重要的是ATP的相對含量，在Pokkali及FR13A都有較IR29高的含量。這些水稻品系特性對於淹水耐受反應的不同，讓我們進一步使用基因微陣列(microarray)去分析各個基因在不同淹水時間點的表現，試圖找出可能調控早期淹水耐受的基因、SUB1A-1所調控的下游基因以及在Pokkali中表現有別於其他兩個品系的基因。我們發現，早期受淹水誘導表現的53個基因中，我們發現了四個轉錄因子與及和鈣離子及MAPK訊息傳導相關的基因；在晚期受淹水誘導的74個基因中，我們發現了被歸類到此群的基因多與醣代謝相關，值得一提的是，我們在這群基因中也發現了這兩個同樣也為第七族的乙烯反應轉錄因子ERF66與ERF67，這與我們實驗室先前的發現結果相符。我們進一步以基因槍轉殖的方式將基因短暫表現於水稻的癒傷組織加引印證，我們發現，ERF66與ERF67受到SUB1A-1的正向調控，此外，ERF66與ERF67可能受到N端法則(N-end rule)所調控，所以，我們推測ERF66、ERF67可能在缺氧穩定後對SUB1A-1有抑制性的回饋機制，微調SUB1A-1對淹水的調控。
另外一方面，在Pokkali中所受淹水誘導表現較高的基因與離子通道、DNA的複製、Expansin及生長有關。而這些基因中，在深水稻曾被報導過會受GA所誘導表現，因此，我們檢查了三種水稻品系中GA代謝相關的基因，結果顯示，使GA從活化態轉換非活化態的基因在Pokkali中有較低的表現，因此我們推測在淹水時，Pokkali中有較多量的GA促使其在淹水時能繼續生長，以利從表層水中得到氧氣。

關鍵字

FR13A ； IR29 ； Pokkali ； 淹水耐受性 ； 激勃素反應

並列摘要

Abstract
Rice SUB1A-1 (SUBMERGENCE1A-1), a member of group-Vll ethylene response transcription factor (ERF-Vll), is up-regulated during submergence, and it plays an important role in flooding tolerance among rice cultivars. The cultivars that contain the SUB1A-1 allele, including FR13A and Pokkali, display flooding-tolerant phenotype. In contrast, IR29 cultivar, which possesses SUB1A-2 allele, shows flooding-intolerant phenotype. In addition, FR13A and Pokkali both contain SUB1A-1 but display significantly different induction patterns and phenotypes under submergence. During submergence, FR13A stays quiescent to conserve energy and its SUB1A-1 transcription is highly induced. On the contrary, Pokkali continues to grow under submergence, but its SUB1A-1 induction level is much lower than that of FR13A. The genes under SUB1A-1 control are mainly involved in glycolytic pathway, which are also highly induced in Pokkali. Moreover, the ATP level is higher in FR13A and Pokkali than IR29 under submergence. Accordingly these materials provided us a good starting point to look for early hypoxia-tolerant genes, downstream genes of SUB1A-1 and specific hypoxia responsive genes in Pokkali. To characterize the tolerance related genes that respond to early and late hypoxia, we applied genome-wide transcriptomic analysis to FR13A, IR29 and Pokkali under submergence. We identified 53 genes that might be involved in the early flooding tolerance. Four transcription factors and genes related to calcium signaling and MAPK pathways were found in this category. On the other hand, 74 genes that might be under SUB1A-1 control in late hypoxia were identified. The genes were mainly related to carbohydrate metabolism. Interestingly, two ERF transcription factors, ERF66 and ERF67, which are potential targets of the N-end rule regulation, were identified in this group. We further used transient assays to validate the regulation in callus. Our results suggested that SUB1A-1 positively regulated ERF66 and ERF67. In addition, ERF66 and ERF67 might follow the N-end rule to modulate SUB1A-1 expression. This regulatory mechanism may fine-tune hypoxia response in rice. On the other hand, the hypoxia responsive genes that were induced higher in Pokkali were associated with ion channel, DNA replication, expansin and growth regulation. Most of them are GA responsive genes and were similarly expressed in deepwater rice under submergence. In addition, the genes encoding enzymes that catalyze the active form GA to inactive form were expressed at lower levels in Pokkali. We proposed that the GA level might be higher in Pokkali which allowed it to continue growing under submergence and get oxygen from water surface.

並列關鍵字

FR13A ； IR29 ； Pokkali ； submergence tolerance ； GA response

參考文獻

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