To explore the regulatory effect of novel miRNA phe-novel-miR276 on lignin synthesis in moso bamboo, so as to provide theoretical reference for material quality improvement and molecular breeding of moso bamboo.

Using bioinformatics methods, we analyzed the novel differentially expressed miRNA (phe-novel-miR276) screened in the preliminary study of moso bamboo. qRT-PCR was applied to detect its expression patterns in different moso bamboo tissues, followed by calculation of relative expression levels and differential analysis. The phe-novel-miR276 was cloned to construct an overexpression plant vector, which was transformed into rice via Agrobacterium mediation for screening positive transgenic lines. Lignin content as well as POD and SOD activities of transgenic and wild-type rice were determined, and intergroup differences of each index were compared and analyzed.

1) Sequence analysis revealed that phe-novel-miR276 shares high homology with the miR398 family, suggesting it is a new member of the miR398 family; 2) phe-novel-miR276 is expressed in the roots, stems, and leaves of P. edulis, with the highest relative expression level in the roots; 3) Phloroglucinol staining showed that the lignin staining depth in the stems and leaves of transgenic lines was lighter; meanwhile, the results of the microdetermination method indicated that the total lignin content in the stems and leaves of transgenic lines was significantly reduced; 4) qRT-PCR detection demonstrated that, compared with wild-type rice, the expression level of Os10g36848-the rice homologous target gene of phe-novel-miR276 (encoding ferulate 5-hydroxylase, F5H)-was significantly downregulated in transgenic lines. Additionally, the POD and SOD activities in transgenic lines were significantly higher than those in wild-type rice, respectively.

In P. edulis, phe-novel-miR276 exerts its regulatory role by downregulating the expression of lignin biosynthesis-related target genes, and indirectly affects the activity of the antioxidant enzyme system in plants.

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.