傷害逆境下受IbHypSys調控表達的甘藷microRNAs之研究

Abstract

植物在遭受到傷害訊號時，會產生胜肽激素以參與植物防禦機制並觸發相關基因的表達，從而保護植物免於受到病原體的入侵等相關逆境。MicroRNA (miRNA) 是由20-24個核苷酸所組成之非編碼RNA，其藉由與互補序列之鹼基形成配對來降低其目標基因的表現。但是，植物胜肽激素和miRNA之間的關係鮮少被研究。本篇研究中，著重於分析甘藷栽培種臺農57號 (Ipomoea batatas cv. Tainung 57) 中，受到甘藷胜肽激素hydroxyproline-rich glycopeptides (HypSys) 所調控的調控miRNA。因甘藷葉片在機械性損傷後會由IbpreproHypSys剪切而成具有功能之IbHypSys。因此，本實驗以過量表達IbpreproHypSys的轉殖株 (OE) 及野生型 (WT) 甘藷，進行small RNA 次世代定序和轉錄組 (transcriptome) 分析。程式預測的方法則是分析系統葉片中OE在受傷2小時前後的miRNA表現量。分析WT與OE之結果顯示出，共有13種miRNA包含miR166、miR398、miR399、miR403和一些新穎miRNA會因受到傷害而下調表現量。綜合比較transcriptome與miRNAs得知，這些miRNAs是會由IbHypSys胜肽進行調控。此外，miRNA的目標基因則是透過psRNAtarget和CleaveLand4兩個不同的分析方式進行預測。因此找出miR398及其目標基因IbSAD2作為後續研究項目。藉由即時定量PCR的結果顯示，在OE的甘藷之中，IbSAD2的表達明顯高於野生型，表示出IbSAD2是受到IbHypSys所誘導而成。並且在菸草中進行agroinfiltration 確認IbSAD2是會受到miR398辨識並切割。綜上所述，甘藷受到傷害逆境後，會促進IbHypSys產生， IbHypSys則會降低miR398的表現，使IbSAD2的表現增加，以進行傷害防禦反應。

Upon wounding, plants produce peptide hormones to engage in plant defense and trigger the expression of defense genes to protect plants from pathogens. MicroRNAs (miRNAs) are non-coding RNA with 20-24 nucleotides and cleave their targets by base pairing with complementary sequences. However, the relationship between plant peptide hormones and miRNAs is seldom investigated. In this study, the regulation of hydroxyproline-rich glycopeptides (HypSys), a plant peptide hormone, on microRNAs were analyzed in sweet potato (Ipomoea batatas cv. Tainung 57). IbHypSys is cleaved from IbpreproHypSys after wounding. The small RNA deep sequencing and transcriptome data from transgenic sweet potato overexpressing IbpreproHypSys (OE) under wounding for 2 hours were analyzed. After these sequencing data from the systemic leaves of wildtype (WT) and OE were compared, 13 miRNAs, comprising of miR166, miR398, miR399, miR403, and some novel miRNAs, were obtained to be down-regulated by wounding. Comprehensive analysis of the transcriptome data and miRNAs suggested that the expression of miRNAs was affected by IbHypSys peptide. Target genes of miRNAs were also predicted by psRNAtarget and CleaveLand4. The miR398 and its target IbSAD2 was further studied. Results of RT-qPCR indicated that the expression of IbSAD2 was significantly increased in OE than in WT, suggesting the expression of IbSAD2 may be induced by IbHypSys. The recognition of miR398 on its binding site on IbSAD2 was further identified by agroinfiltration, indicating IbSAD2 was a target of miR398. The expression of target gene IbSAD2 might enhance the response of plants to wounding. In conclusion, IbHypSys-induced miRNAs may regulate wounding response, and this research may illuminate wounding defense in sweet potato.