探討microRNA在甘薯傷害的防禦機制

Study of microRNAs involved in wounding response in sweet potato

林振祥(Jeng-Shane Lin)

指導教授 ： 鄭石通

國立臺灣大學/生命科學院/植物科學研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.02041

摘要

microRNA (miRNA)是一段由生物體內自行合成約21-24鹼基的小分子核醣核酸，不轉譯出任何蛋白，其藉由降解mRNA或抑制mRNA進行蛋白質轉譯來達成調控基因表現。近年來在阿拉伯芥上發現超過百種的miRNA，且研究指出miRNA參與植物不同階段的生長發育及抵抗不同環境的變化，所以我們對於與植物傷害相關的miRNA有著極大興趣，希望探討傷害相關miRNA的訊息傳遞的過程。藉由miRNA序列在演化具有高度保留性的特性，利用阿拉伯芥miRNA的序列當作探針探討甘藷植株在遭受傷害後miR159、miR407、miR828的表現情形。在甘藷植株中無偵測到miR407的表現；miR159可在莖中被偵測到，而在葉中表現量極低；miR828則在葉中被偵測到。此外，在甘藷傷害後，miR159表現量並未隨時間不同有所變化，而miR828表現量卻會被抑制。且處理誘導傷害防禦機制相關的分子，如:過氧化氫(hydrogen peroxide, H2O2)、一氧化氮(nitric oxide, NO)、甲基茉莉酸(Methyl jasmonate, MeJA)、離層酸(abscisic acid, ABA)及乙烯(ethylene)，也會抑制miR828的表現。我們再藉由不同誘導劑與抑制劑探討miR828表現情形進而探討其訊息傳遞的過程，推測植物傷害後，促使NO與MeJA生合成，而MeJA也會而活化NOS產生NO，而NO再活化NADPH oxidase產生H2O2，或者MeJA本身也會透過過其他分子，而抑制miR828表現進而調控miR828標的基因表現。此外，ethylene也會透過MeJA抑制miR828表現，而一氧化碳(carbon monoxide, CO)會在之中清除H2O2進而抑制傳遞過程。而miRNA藉由序列互補性辨識標的基因，進而對標的基因進行靜默 (silencing)。我們利用cDNA clone釣取到三個可能的miR828標的基因，分別為receptor protein kinase (RPK)、TLD和MYB。藉由傷害後因miR828表現被抑制而減少對標的基因mRNA降解或降低抑制標的基因蛋白質轉譯，使得標的基因的表現進而參與傷害防禦調控機制。

關鍵字

甘薯 ； 傷害 ； microRNA ； 標的基因 ； 靜默

並列摘要

MicroRNAs (miRNAs) contain approximate 22 nucleotides that post-transcriptionally regulate gene expression by directing mRNA cleavage or translational inhibition. Currently, more than one hundred miRNAs that play multiple roles in growth, development, and stress responses of plants have been identified in Arabidopsis thaliana. Here, I investigated the expression patterns of miR159, miR407, and miRNA828 upon wounding in sweet potato. The expression of miR407 was undetectable in leaves and stems. However, miR159 was expressed in stems and, at low levels, in leaves, whereas miR828 was expressed in leaves and undetectable in stems. Furthermore, the expression of miR828 was decreased upon wounding in sweet potato. Additionally, several defensive responding compounds, including hydrogen peroxide (H2O2), nitric oxide (NO), methyl jasmonate (MeJA), abscisic acid (ABA), and ethylene also suppressed miR828 expression. However, carbon monoxide (CO) suppressed repression of miR828 by wounding. Interactions among these defensive responding compounds for the regulation of miR828 were also studied. Results suggested that wounding stimulates the biosynthesis of MeJA and NO. Then, MeJA activates NO synthase to generate NO. Finally, NO inspires the activity of NADPH oxidase to generate H2O2, which suppress miR828 expression. In addition to miR828, three potential miR828 targeted genes, receptor protein kinase (RPK), TLD, and MYB were isolated and were up-regulated by wounding. In addition, RPK, TLD, and MYB were regulated by NO and H2O2 when miR828 was repressed. These results imply that wounding represses the expression of miR828, and consequently regulates the expression of RPK, TLD, and MYB genes to protect plants from damage.

並列關鍵字

microRNA ； miR828 ； sweet potato ； target gene ； wounding

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