甘藷中受一氧化碳調控之嶄新miRNA-tag202調控 MAPKKK-like protein

Abstract

一氧化碳是一種二次訊息傳遞氣體分子，在生物體的生長發育以及免疫反應中皆扮演著重要的角色。microRNA (miRNA)是一群21至24個核苷酸長、不轉譯出蛋白質的小分子核醣核酸，藉由降解mRNA或抑制mRNA轉譯的方式，來調控目標基因的表現量。然而，很少研究指出miRNA會受到一氧化碳的影響。本篇研究藉由次世代定序技術建構一氧化碳處理以及對照組的甘藷葉small RNA定序(small RNA sequencing)資料庫，和未經任何處理的甘藷葉轉錄體資料庫(transcriptome)，交互比對分析資料庫後預測會受一氧化碳影響的一群miRNA candidates，以及這些miRNA candidates所調控的目標基因，並進行後續實驗的驗證。結果顯示，一條長度為22個核苷酸、暫名為tag202的新穎miRNA會受一氧化碳所誘導。在菸草中同時短暫表現precursor form tag202 (pre-tag202)與其預測目標基因的序列互補位置(Binding Site-GFP，BS-GFP)，證實BS-GFP會被tag202所剪切。而在大量表現與降低表現tag202的甘藷轉殖株中，確認目標基因的mRNA累積量確實與tag202的表現量呈相反趨勢。除此之外，經由快速擴增cDNA末端(RACE)調取目標基因全長，推測此目標基因應為MAPKKK類似蛋白。因此，在甘藷中會受到一氧化碳誘導的新穎miRNA tag202，很可能在MAPK訊息傳遞路徑中，扮演著重要的角色。

Carbon monoxide, a gaseous secondary messenger, plays an important role in growth and immunity responses. MicroRNAs (miRNA), a group of 21-24 nucleotides small noncoding RNA, direct messenger RNA cleavage or inhibit translation to regulate gene expression. However, few studies demonstrated miRNAs are affected by CO. In this study, next generation sequencing (NGS) was used to generate two small RNA sequence databases of sweet potato (Ipomoea batatas cv. Tainung 57) leaves with or without CO treatment, and one transcriptome of sweet potato leaf without any treatment. Those databases were comparatively analyzed to predict the small RNAs affected by CO, and the target genes regulated by small RNAs. Then, their interactions and physiological functions were further validated. Results showed that a CO-induced 22-nucleotide-long novel miRNA temporarily named tag202 was identified. The interaction between tag202 and potential target gene binding site fused GFP (BS-GFP) was confirmed by agro-infiltration. Sweet potatoes of tag202 overexpression line and knock-down line were created, and the expressions of predicted target gene showed the opposite trend of those of tag202’s in these transgenic plants. Moreover, the target gene was found that it may encode a MAPKKK-like protein by rapid amplification cDNA ends (RACE). Conclusively, tag202 affected by CO may function as a switch to regulate mitogen activated protein kinase (MAPK) pathway in plants.