文心蘭乙烯訊息傳導途徑中小核糖核酸與其目標基因之辨識

Abstract

文心蘭 (Oncidesa) 是富有經濟價值的切花，其花部老化受乙烯影響。藉由RNA干擾技術默化乙烯訊息傳導途徑中關鍵的轉錄因子ETHYLENE INSENSITIVE3 (EIN3) 基因具有延長花期的效果。為瞭解默化EIN3對其他基因表現的影響與小核糖核酸 (small RNA, sRNA) 的調控機制，本研究分析文心蘭南西OgEIL1默化轉殖株與未轉殖株間轉錄體 (transcriptome) 與 sRNA資料庫的表現差異，嘗試建構OgEIL1在文心蘭中的調控網絡。經高通量定序葉片與花部轉錄體後，以de novo組裝法組裝出587,771條轉錄本 (transcripts)。在sRNA資料庫中，葉片比對出177條已知的miRNA，並預測出8條新的miRNA，花部中則比對出31條已知miRNA與20條新的miRNA。從轉錄本與miRNA的差異表現分析 (differentially expressed analysis) 得知，葉與花部位間的差異表現基因比OgEIL1默化轉殖株與未轉殖株間差異表現基因多。經基因本體 (gene ontology) 註解富集檢定 (enrichment test) ，OgEIL1默化轉殖株中相對未轉殖株表現量較高的序列多和硫代謝相關。OgEIL1默化轉殖株與未轉殖株於葉片和花部共比對出52與9條差異表現miRNA，其中miR395a與miR408在OgEIL1默化轉殖株葉片與花部中表現量皆較未轉殖株高，而miR156a、miR157d與miR396a則在兩部位皆較未轉殖株低。此外，受EIN3負向調控的miR164家族基因表現分析，顯示在OgEIL1默化轉殖株葉片中，miR164a、miR164b與miR164c有上調的情形。經預測文心蘭差異表現miRNA於轉錄體中的作用目標基因為EIL、NAC、AP2等與乙烯相關的轉錄因子。

Oncidesa is a popular orchid with high economic value in cut-flower markets around the world. Ethylene involves petal senescence of Oncidesa. When ETHYLENE INSENSTIVE3 (EIN3), the key transcriptional factor in ethylene signal transduction, was knocked down by gene silencing, the shelf-life of Oncidesa had been prolonged. To understand gene expression and regulation of small RNA, we analyzed transcriptomes of OgEIL1 RNA interfering transgenic and non-transgenic plant, and tried to construct the OgEIL1 regulatory network in Oncidesa. By high-throughput sequencing and de novo assembling, we got 587,771 transcripts in Oncidesa transcriptome. Among the sRNA sequence of Oncidesa, a total of 177 and 31 known miRNAs were annotated in leaf and flower, respectively. After predicted by software, 8 and 20 putative novel miRNAs were identified in leaf and flower, respectively. There are more differentially expressed transcripts and miRNAs between organs than between transgenic and non-transgenic plant. According to gene ontology enrichment test of differentially expression transcipts in OgEIL1 RNA interfering plant, many enriched GO terms were related to sulfur metabolic process. Comparison of the miRNAs expression level between the transgenic plant and non-transgenic Oncidesa revealed that miR395a and miR408 were up-regulated and miR156a, miR157a, miR157d and, miR396a were down-regulated in both organs. Tthe expression level of miR164 family genes which are negatively regulated were analyzed. The data showed that miR164a, miR164b, and miR164c-5p were up-regulation in the OgEIL1 RNA interfering transgenic plant. The target transcripts of differentially expressed miRNAs were identified and some of them are transcription factors involving in ethylene response, such as EIL, NAC and AP2.