ACC氧化酶基因默化轉殖香蕉果實之微RNA (microRNA) 分析

香蕉是一種典型的更年性果實，後熟期間會有呼吸速率突然上升、伴隨乙烯生合成之反應，造成果實快速老化，縮短櫥架壽命。ACC氧化酶 (ACC oxidase, ACO) 為催化乙烯生合成最終步驟的酵素。利用農桿菌媒介法轉殖以及RNA干擾 (RNA interference, RNAi) 技術，分別將兩個香蕉ACC氧化酶基因Mh-ACO1及Mh-ACO2默化，得到的轉殖株之果實後熟皆受到延遲。以此二轉殖株後熟第三期的果實為材料，對其內微RNA (microRNA, miRNA) 進行次世代定序，再與轉錄體結果進行整合分析，結果預測的miRNA靶基因主要富集在KEGG之硫傳遞系統、蛋白質消化吸收、组胺酸代谢、致病性大腸桿菌感染、硫代谢五條途徑。經即時定量反轉錄聚合酶連鎖反應 (qRT-PCR) 分析香蕉果實後熟過程的基因表現，結果隨著果實後熟度增加，miRNA表現量下降，靶基因的表現量則上升，顯示miRNA藉由調控靶基因表現，負向調控香蕉果實後熟。

基因默化香蕉轉殖株中miRNA基因差異表現之聚類分析

基因默化香蕉轉殖株中miRNA與預測之靶基因關係圖

Xia, Y., Z. Lai, Y.-Y. Do, P.-L. Huang. 2023. Characterization of microRNAs and gene expression in ACC oxidase RNA interference-based transgenic bananas. Plants 12:3414. (20 pages) doi:10.3390/plants12193414. (SCI)

研究全文：https://doi.org/10.3390/plants12193414

Characterization of MicroRNAs and Gene Expression in ACC Oxidase RNA Interference-Based Transgenic Bananas

Banana (Musa acuminata, AAA group) is a typically respiratory climacteric fruit. Previously, genes encoding ACC oxidase, one of the key enzymes in ethylene biosynthesis, Mh-ACO1 and Mh-ACO2 in bananas were silenced individually using RNAi interference technology, and fruit ripening of transgenic bananas was postponed. Here, the differential expression of miRNAs and their targeted mRNAs were analyzed in the transcriptomes of fruits at the third ripening stage, peel color more green than yellow, from the untransformed and RNAi transgenic bananas. Five significantly differentially expressed miRNAs (mac-miR169a, mac-miR319c-3p, mac-miR171a, mac-miR156e-5p, and mac-miR164a-5p) were identified. The predicted miRNA target genes were mainly enriched in six KEGG pathways, including ‘sulfur relay system’, ‘protein digestion and absorption’, ‘histidine metabolism’, ‘pathogenic E. coli infection’, ‘sulfur metabolism’, and ‘starch and sucrose metabolism’. After ethylene treatment, the expression of ACC oxidase silencing-associated miRNAs was down-regulated, and that of their target genes was up-regulated along with fruit ripening. The evolutionary clustering relationships of miRNA precursors among 12 gene families related to fruit ripening were analyzed. The corresponding expression patterns of mature bodies were mainly concentrated in flowers, fruits, and leaves. Our results indicated that ethylene biosynthesis is associated with miRNAs regulating the expression of sulfur metabolism-related genes in bananas.

Cluster analysis of differentially expressed miRNAs in small RNA sequencing of RNAi transgenic banana

Relationships between miRNAs and their targeted transcription factors

Xia, Y., Z. Lai, Y.-Y. Do, P.-L. Huang. 2023. Characterization of microRNAs and gene expression in ACC oxidase RNA interference-based transgenic bananas. Plants 12:3414. (20 pages) doi:10.3390/plants12193414. (SCI)

More information: https://doi.org/10.3390/plants12193414