In Silico Search and Biological Validation of MicroR171 Family Related to Abiotic Stress Response in Mulberry (Morus alba)

ABSTRACT miR171 belongs to a highly conserved and ubiquitously expressed microRNA gene family across species that play a critical role in controlling plant growth and development through the regulation of the miR171-SCL (scarecrow-like proteins) module. There is limited research available on the evolutionary relationship and functional diversification of miR171 members. In this study, we identified eight miR171 genes in the mulberry genome by bioinformatics analysis that were subsequently used to compare the evolutionary levels and explore abiotic stress mechanisms mediated by mno-miR171s (Morus notabilis miR171s). The results of phylogenetic analysis showed that the mature mno-miR171 sequences have strong sequence conservation, but their critical sites also exhibit high variation leading to functional diversification. Through quantitative real-time PCR, the expression profile of each mno-miR171 was analyzed under different stress treatments. All mno-miR171s, apart from mno-miR171h, were found to be significantly up-regulated under salt and drought stress conditions. The target genes of mno-miR171a namely, Morus020885 and Morus011800, were predicted and verified using the plural RNA method. 5′-rapid amplification of complementary DNA ends assays further to reveal that the target genes could be degraded by mno-miR171a post-transcriptionally. Overexpression of mno-miR171a in Arabidopsis improved the percentage of seed germination when the seeds were grown in NaCl- and mannitol-containing media. Transgenic plants were observed to grow better under drought conditions. The expression of various stress genes was significantly higher in transgenic plants than in wild type, except ERF11. Taken together, our study confirmed that mno-miR171a enhanced plant resistance to adverse stress environments via the regulation of the SCL targets.