探討甘藷 Small RNAs 在傷害後的反應機制

Abstract

在植物中，Small RNAs (sRNAs) 是 20-24 個核苷酸之內生性、普遍存在的小片段 RNA，參與在轉錄基因靜默 (transcriptional gene silencing (TGS)) 和轉錄後基因靜默 (posttranscriptional gene silencing (PTGS))，並主要分為兩大類：microRNAs (miRNAs) 和 small interfering RNAs (siRNAs)，但其參與在傷害反應的功能研究不多；此外，次級 siRNAs (secondary siRNAs) 中的一個分類 trans-acting small interfering RNAs (tasiRNAs)，其觸發者 (trigger) 多為一段 22 個核苷酸的 miRNA，致使產生多段 21 個核苷酸的 tasiRNAs。 為了瞭解甘藷 (sweet potato (Ipomoea batatas cv. Tainung 57)) sRNAs 在傷害反應下所扮演的角色，以傷害成熟葉片產生深度定序 (deep sequencing) 資料庫，透過程式設定條件進行篩選，篩去約 1.6*107 筆資訊，並將結果與 Rfam 資料庫進行比對刪去，再以即時定量聚合酶連鎖反應 (real-time PCRs) 確認傷害後表現趨勢，選取了兩個皆受傷害所抑制的 sRNAs：S4 與 S5。從實驗室甘藷轉錄組 (transcriptome) 資料庫選取 S4 的推定目標基因 84637 和 79917，S5的推定目標基因 266860，且這些目標基因的確在傷害後累積，與 sRNAs 表現趨勢呈現負相關。同時也發現了前驅物 (precursor)：72855a 和 72855a.2，能產生類似 S4 和 S5 tasiRNAs，而其觸發者為 22 個核苷酸的保守性 miR2118 家族成員：miR2118a，miR2118a.2 和 miR2118a.3。 甘藷葉片處理傷害後之傷害訊息 (wounding signaling) 雙路徑被建立，路徑之一為透過抑制 S4 和 S5 siRNAs，進一步使目標基因：84637，79917 和 266860 累積。另一條路徑為透過抑制 miR2118 家族成員共同的前驅物 RC-comp122014，與抑制 tasiRNA 前驅物：72855a 和 72855a.2，使得類似 S4 和 S5 tasiRNAs 無法產生，進而累積可能的目標基因：84637-LIKE，79917-LIKE 和 266860-LIKE，或是其他傷害相關基因，藉由雙路徑可全面性地使傷害相關基因累積，提升對傷害反應的效率。

Small RNAs (sRNAs), 20-24 nt in length, are endogenous and responsible for transcriptional gene silencing (TGS) and posttranscriptional gene silencing (PTGS) in plants. They are ubiquitous and mainly classified into two groups, microRNAs (miRNAs) and small interfering RNAs (siRNAs), but limited research has been focused on their functions in wounding responses. Additionally, the trans-acting small interfering RNAs (tasiRNAs) are a class of secondary siRNAs and are triggered by 22-nt miRNA to produce 21-nt phased patterns. To address the roles of novel sRNAs involved in wounding responses in sweet potato (Ipomoea batatas cv. Tainung 57), a large deep sequencing data from wounded mature leaves were generated. Approximately 1.6*107 records were screened through the conditional analysis software. After the analysis in the Rfam, real-time PCRs were performed to validate the expression of sRNAs in sweet potato upon wounding. Two small RNAs, S4 and S5, which are repressed by wounding, were chosen for further study. In addition, the putative target genes 84637 and 79917 for S4, and gene 266860 for S5 were selected from the Ipomoea batatas-transcriptome database. The three target genes for S4 and S5 were accumulated by wounding and a negative correlation of expression between target gene and small RNA existed. The precursors of both S4-like and S5-like tasiRNAs, 72855a and 72855a.2, were identified. 22-nt members of conserved miR2118 family, miR2118a, miR2118a.2 and miR2118a.3, trigger the production of S4-like and S5-like tasiRNAs. A wounding bi-pathway which siRNAs participate in was built, sweet potato leaves responded to wounding stress through repressing S4 and S5 siRNAs to accumulate the expression levels of target genes, 84637, 79917 and 266860. Moreover, suppressing miR2118 family precursor (RC-comp122014) and tasiRNAs precursors (72855a and 72855a.2) is the other way to repress the production of S4-like and S5-like tasiRNAs in order to accumulate the expression levels of 84637-LIKE, 79917-LIKE and 266860-LIKE genes, or other target genes. This wounding bi-pathway can comprehensively accumulate the gene families or other wounding-related genes expression levels to enhance the efficiency.