利用生物資訊軟體在甘藷中探索傷害逆境相關之嶄新miRNAs

Abstract

植物防禦機制基因受到轉錄、轉錄後以及轉譯階段的調控。MicroRNA (miRNA)在傷害反應的轉錄及轉錄後調控扮演著重要的角色。為了研究甘藷傷害反應的miRNA，我們實驗室先前建構small RNA libraries，處理組別為對照組與傷害處理後30分鐘。由於甘藷不是已解序物種，我們也建構了甘藷的de novo transcriptome作為處理small RNA deep-sequencing data序列比對時的參考序列。我們利用次世代定序技術進行定序，得到大量的序列資料。實驗室前人朱敏瑤利用miRBase資料庫比對分析，發現許多傷害相關的conserved miRNA。然而，隨著演化產生差異，不同物種間應存在許多不保守性的novel miRNA參與植物生理反應。本實驗利用miRNA生合成過程中的兩個特性─stem-loop structure之precursor和miRNA/miRNA* duplex，開發Linux軟體miRcutter以預測甘藷傷害反應下novel miRNA。藉由定序資料比對分析，我們找到128條novel miRNA precursor以及16條conserved miRNA precursor。我們以small RNA定序資料中傷害前後表現量差異大作為篩選條件，挑選出5個novel miRNA precursor，暫名為pre1-5。利用反轉錄PCR (RT-PCR)檢測pre1-5在傷害處理下的表現量。結果顯示pre1-5皆會受傷害調控，並且其傷害30分鐘後的表現量，與pre1-5在small RNA定序資料預測之趨勢一致。利用miRNA目標基因預測軟體Mir_score，我們預測了novel miRNA的目標基因。Novel miRNA tag-292在傷害反應中可能藉由調控putative auxin efflux carrier component 8-like (AC8)的基因表現量來改變auxin濃度。本實驗發現甘藷傷害反應的novel miRNA precursor，並透過預測novel miRNA和目標基因的調控關係，更了解甘藷傷害反應的全貌。

Subjected to wounding stress, plants regulate genes related to many physiological processes. It was known that plant hormones and secondary metabolites are associated with wounding responses. However, much less is known about post-transcriptional controls in wounding responses. MicroRNAs are a class of noncoding RNAs, which typically function by guiding ARGONAUTE to cleave or block target mRNAs. Thus, miRNAs play a crucial and currently uncertain part in regulating post-transcriptional gene expression in wounding response. In previous studies, we constructed small RNA deep-sequencing libraries with wounding and its control treatments in sweet potato (Ipomoea batatas L.). We also constructed the de novo transcriptome in sweet potato as reference sequences. The miRcutter, a software program under Linux, was developed to map small RNA deep-sequencing data into the transcriptome for identification of novel miRNAs precursors and miRNA*. By miRcutter, 128 novel miRNA precursors and 16 conserved miRNA precursors were found. Further, the expression levels of 5 novel miRNA precursors, pre1-5, were confirmed to correspond with those of deep sequencing data in wounding responses. A gene which is annotated as an auxin efflux carrier component is one of the interesting target genes recognized by tag-292, a novel miRNA, in wounding responses.