MicroRNAs (miRNAs) 是一類長約21個核苷酸的單鏈小分子RNA，lin-4是最早於線蟲 (Caenorhabditis elegans) 中首次發現的miRNA，目前miRNAs在許多生物中已被發現。miRNAs和目標基因結合後經由轉錄抑制或mRNA降解等方式調控基因表現。植物體中，miRNAs主要參與生長發育、荷爾蒙和逆境等調控。阿拉伯芥中已知miR156和miR172兩個 miRNAs參與在生長時期轉換和開花時期調控，從營養生長進入生殖生長時期，miR156表現逐漸下降，其目標基因SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL)因而增加表現，SPL進而促進miR172表現，其目標基因APETALA2 (AP2) 和AP2-like 基因, TARGET OF EAT 1 (TOE1), TOE2受到抑制因而誘導開花。本實驗以白花蝴蝶蘭 (Phalaenopsis aphrodite subsp. formosana) 為材料，白花蝴蝶蘭為台灣重要原生種具有多花色、獨特花型且具高觀賞價值，但因其生長週期長，所以本篇藉由探討microRNAs (miR156和miR172) 在白花蝴蝶蘭開花調控所扮演的角色，未來希望能縮短蝴蝶蘭開花所需時間。我們利用小分子RNA定序技術並配合生物資訊分析和其他物種已知miRNAs比對後得知蝴蝶蘭有22個miRNA families，當中包含miR156 和miR172。並利用小分子RNA北方點墨法證實 miR156 和miR172確實存在蝴蝶蘭，且miR156在葉片表現高於梗和花苞，miR172則是梗和花苞表現較高。進一步由實驗室所建立的蝴蝶蘭transcriptome databaes中找尋可能的目標基因，PaSPL和PaAP2 分別具有可被miR156 和miR172所結合之位置且皆會被cleavage。其中PaSPL於RNA層次上在不同組織的表現和 miR156 互相互補，推測miR156具有調控PaSPL的功能 ; PaAP2在RNA層次之表現上並沒有因miR172表現低而增加。本實驗結果顯示蝴蝶蘭具有植物間高保守性的miRNAs且存在經由miRNAs所調控的開花路徑。

MicroRNAs (miRNAs) are ~21-nucleotide noncoding RNAs that have been identified in various organisms. In plants, miRNAs are mostly involved in the regulation of important growth and developmental processes such as leaf and root development, phase transitions, and flowering. Recent studies explore two temporally expressed miRNAs in Arabidopsis, miR156 and miR172 play important roles in regulation of vegetative phase change and flowering time regulation. From juvenile-to-adult transition, the transcription of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) accumulation depend on the decrease of miR156 with time, and SPL genes promote miR172 transcript which then repress the APETALA2 (AP2), and AP2-like genes、TARGET OF EAT 1 (TOE1)、TOE2 to induce flowering. In this study, we used Phalaenopsis aphrodite subsp. formosana as material. To exam the possibility of controlling miR156 and miR172 expression in this long life cycle plant to accelerate the flowering time, we perform small RNA sequencing analysis and combining bioinformatics, we discovered 22 conserved miRNA families, and identified miR156 and miR172 othologs from Phalaenopsis orchid. Also, we discovered miR156 expresses higher in leaves than in stalks and in flower buds; however miR172 mainly expressed in stalks. These patterns are similar with those in Arabidopsis, imply us that we have possibility to overexpress miR172 to facilitate flowering in orchid the same way as Arabidopsis. Further, we identified the target genes of miR156 and miR172 from our orchid transcriptome databaes, PaSPL and PaAP2 which contain a miR156 and miR172 complementary site. The expression of PaSPL is abundant in stalks and flower buds that is complementary with expression of miR156. This suggests that miRNA-mediated flowering pathway exist in Phalaenopsis orchid.

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