鑑定日日春微型核酸及其調控基因受花生簇葉病菌質體感染所導致花器葉片化之研究

花生簇葉病菌質體（Peanut Witches’ Broom Phytoplasma, PnWB）會對日日春的花部器官造成花瓣綠化及花器葉化等病徵。為從 RNA 層級著手探討花器葉化之致病機制，我們將日日春健康花（HF）及感病第四期花（S4）樣本之轉錄體及小分子核糖核酸（small RNA）以次世代解序技術（next-generation sequence, NGS）進行解序，並建立資料庫以利後續開花相關基因之鑑定及微型核糖核酸（miRNA）對基因表現調控之研究。本研究以實驗策略來分析經次世代解序技術解序之轉錄組及 small RNA，進而鑑定出非模式植物日日春之保守性 miRNA 及其目標基因。流程之建立立基於諸多生物性特性，包括：miRNA/miRNA* 之配對特性、miRNA/miRNA* 坐落於其前驅體二級結構上之位置特性、miRNA/miRNA* 累積量之比例特性及 miRNA reads 於前驅體上之分布特性等。另外，miRNA-target 計分原則及 miRNA 及其目標基因相反表現趨勢之特性亦被用於優化日日春中 miRNA 目標基因之預測。本研究預測出 23 個保守性 miRNA 及 71 個可能受 miRNA 調控之目標基因。據結果，大部分 miRNA 之表現在 S4 中下降，故提出花生簇葉病菌質體可能在感染過程中抑制 miRNA 之生合成之推論。此外，由本研究預測出之 miR396 及其目標基因 SHORT VEGETATIVE PHASE (SVP) 間之配對關係而推論由 FLOWERING LOCUS T (FT) 調控之開花路徑可能受到PnWB植物菌質體調控而導致花器葉片化病徵之產生。

關鍵字

花生簇葉病菌質體；次世代解序技術；微型核酸預測；微型核酸目標基因預測；花器葉片化

並列摘要

Peanut Witches’ Broom (PnWB) Phytoplasma causes floral symptoms such as virescent petal and phyllody on an indicator plant periwinkle Cantharanthus roseus. To unravel the reprogramming mechanism underlying the symptom development in RNA level, the deep sequenced transcriptome and small RNA profile of healthy (HF) and diseased stage-4 (S4) flower were established for studying flowering-related gene expression and miRNA-mediated gene regulation. The experimental approach was used to analyze the transcriptome and small RNA profiles to identify the conserved microRNA (miRNA) and their targets in periwinkle. The pipeline was developed based on several biological features, e.g. miRNA/miRNA* paring, miRNA/miRNA* duplex location on precursor, ratio of reads between miRNA and miRNA*, and miRNA reads distribution on precursor etc., to predict the miRNA of periwinkle. In additon, miRNA-target score, and the opposite expression tendency between miRNA and transcriptome were also considered in the prediction pipeline for enhancing the accuracy of target prediction. A total of 23 conserved miRNAs and 71 targets of periwinkle were predicted in this study. The results showed that most Cr_miRNAs were decreased in S4 sample and implied that PnWB might suppress these miRNAs during infection. The predicted relation of Cr_miR396 and its targets (Cr_SVP1 and Cr_SVP2) was highlighted in involvement of Cr_FT mediated flowering pathway between HF and S4 samples, and therefore the mechanism of the leafy flower reprogramming was proposed in this study.

並列關鍵字

PnWB phytoplasma ； NGS ； miRNA prediction ； miRNA target prediction ； leafy flower

參考文獻

Yang, I.-L. (1985). Host Responses of Peanut Witches' Broom Disease, Journal of Agricultural Research of China 34, 464-468.

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Alvarez-Buylla, E. R., Pelaz, S., Liljegren, S. J., Gold, S. E., Burgeff, C., Ditta, G. S., Ribas de Pouplana, L., Martinez-Castilla, L., and Yanofsky, M. F. (2000). An ancestral MADS-box gene duplication occurred before the divergence of plants and animals. Proc Natl Acad Sci U S A 97, 5328-33.

Aufsatz, W., Mette, M. F., van der Winden, J., Matzke, A. J., and Matzke, M. (2002). RNA-directed DNA methylation in Arabidopsis. Proc Natl Acad Sci U S A 99 Suppl 4, 16499-506.

Bertaccini, A. (2007). Phytoplasmas: diversity, taxonomy, and epidemiology. Front Biosci 12, 673-89.

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鑑定日日春微型核酸及其調控基因受花生簇葉病菌質體感染所導致花器葉片化之研究

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