甘藷受傷後系統葉中的嶄新miRNA-tag6568的研究

Abstract

植物在外界環境下，會受到生物及非生物等逆境。其中，傷害逆境對植物影響最為嚴重，妨礙了植物的生長及發育。而植物也已經演化出許多防禦機制來因應傷害逆境，包含修復受傷組織，及活化系統性防禦機制，使尚未受到傷害之部位產生防禦，因應即將到來之逆境。植物的microRNA (miRNA)由21至24個核苷酸所組成的非編碼小片段分子，藉由降解目標基因之mRNA或抑制轉譯的方式，來調控目標基因。然而，目前對於參與在甘藷系統性防禦之miRNA了解甚少。本篇研究藉由次世代定序技術建構傷害處理組以及對照組的甘藷系統葉small RNA定序(small RNA sequencing)資料庫，和未經處理的甘藷系統葉轉錄體資料庫(transcriptome)，交互比對資料庫後，預測會受傷害影響的一群miRNA candidates，以及這些miRNA candidates所調控的目標基因。分析發現，有319個miRNA可能參與系統葉防禦反應，其中131個為已知的miRNA，188個為新穎的miRNA。我們挑選了長度為21個核苷酸的新穎miRNA，tag6568。利用甘藷系統葉轉錄體資料庫(transcriptome)預測出tag6568的目標基因可能為LRR receptor-like protein kinase 1 (RPK1) 和CDPK-related kinase 1 (CRK1)。經即時定量PCR分析後primary form tag6568和mature form tag6568在系統葉的表現量會受傷害誘導，而其目標基因RPK1和CRK1表現量會受傷害抑制。然而在菸草短暫基因表現實驗中，結果顯示RPK1和CRK1表現量不受tag6568調控，這些結果表明RPK1和CRK1是由生物資訊預測出tag6568的目標基因，然而他們的相互作用和生理功能將進一步研究。

Wounding stress is the most serious impact on plants, which affects plant growth and development. Plants have evolved many defense mechanisms to deal with damage, including repairing injured tissues and activating systemic defense mechanisms. Plant microRNAs (miRNAs), a group of 21-24 nucleotides small non-coding RNA, direct messenger RNA cleavage or inhibition translation to regulate gene expression. Previous studies show that miRNAs may involve in local wounding. However, little is known about the miRNAs involved in the systematic defense of sweet potatoes. In this study, next generation sequencing (NGS) was used to generate the transcriptome and two small RNA sequence databases of sweet potato (Ipomoea batatas cv. Tainung 57) systemic leaves with or without wounding treatment. These databases were comparatively analyzed to predict the small RNAs affected by systematic wounding, and target genes regulated by small RNAs. Here we find 319 miRNA candidates involve in the wounding of the sweet potato systemic leaves, 131 of them are conserved miRNAs, and 188 of them are novel miRNAs. We focus on a wounding-induced 21-nutleotide-long novel miRNA named tag6568. Target gene search based on sweet potato transcriptome database, we find that LRR receptor-like protein kinase 1(RPK1) and CDPK-related kinase 1 (CRK1)may be the putative target. Verified by quantitative-RT PCR, expression of primary form tag6568 and mature form tag6568 are both increased after wounding. Conversely, the expression ofRPK1and CRK1 were decreased. These result indicated that RPK1 and CRK1 may regulate by tag6568. However, the results show that RPK1 and CRK1aren’t targrted by tag6568 inTransient expression assay by agro-infiltration.These results indicate that RPK1 and CRK1 are predicted by bioinformatics to target genes of tag6568, however their interactions and physiological functions will be further studied.