Identification and differential analysis of miRNAs associated with cell wall formation in Phyllostachys edulis under drought stress

Through high-throughput transcriptome sequencing of seedlings of Phyllostachys edulis under 20% PEG 6000 drought stress at different time periods (0, 6, 12, 24, 48 h), we investigated the differential miRNAs that regulate the cell wall formation in response to the drought stress, and screened for the differential miRNAs and their target genes. This study aims to provide an important theoretical basis for the adaptive evolution of bamboo in terms of cell wall, as well as potential candidate gene resources for molecular breeding of bamboo.

Differential analysis of drought-treated moso bamboo leaves was carried out by bioinformatics, and the expression patterns and targeting roles were verified by real-time fluorescence quantification and dual-luciferase assay.

408 miRNAs were identified in 15 samples, including 51 known miRNAs, 357 newly predicted miRNAs and 7 105 predicted target genes. A total of 52 differentially expressed miRNAs were found under drought stress, and three differentially expressed miRNAs were unearthed: novel_miR116, novel_miR276 and novel_miR2, corresponding to target genes enriched on the Kyoto Encyclopedia of the Genome (KEGG) to pathways closely related to cell wall formation; PH02Gene11396 encodes steroid 5-α-reductase (Det2), PH02Gene36673, PH02Gene36674, and PH02Gene21400 encode ferulic acid-5-hydroxylase (F5H); and PH02Gene50651 encodes growth hormone-responsive protein (AUX/IAA); the overall expression of the target miRNA decreased with the deepening of drought stress, while the expression of the target gene increased with the deepening of drought stress; the dual luciferase activity of the target miRNA mixed with the target gene was significantly lower than that of the miRNA empty vector mixed with the target gene, and the fluorescence intensity of the right injected miRNA-mRNA was significantly lower than the fluorescence intensity of the left injected miRNA empty vector-mRNA on the same tobacco leaf.

Three target miRNAs and four target genes were screened in this study, revealing the following negative regulatory targeting relationships: novel_miR116 with PH02Gene11396, novel_miR276 with PH02Gene36673/PH02Gene36674, novel_miR2 with PH02Gene50651. these target miRNAs may affect cell wall formation by regulating the expression of oleoresin lactones, lignin monomers, and growth hormone response proteins.