Identification and Expression Analysis of miR160 and Their Target Genes in Cucumber.

miR160 plays a crucial role in various biological processes by regulating their target gene auxin response factor (ARF) in plants. However, little is known about miR160 and ARF in cucumber fruit expansion. Here, 4 Csa-MIR160 family members and 17 CsARFs were identified through a genome-wide search. Csa-miR160 showed a closer relationship with those in melon. Phylogenetic analysis revealed that CsARFs were divided into four classes and most of CsARFs presented a closer evolutionary relationship with those from tomato. Putative cis-elements analysis predicted that Csa-MIR160 and CsARFs were involved in light, phytohormone and stress response, which proved that they might take part in light, phytohormone and stress signaling pathway by the miR160-ARF module. In addition, CsARF5, CsARF11, CsARF13 and CsARF14 were predicted as the target genes of Csa-miR160. qRT-PCR revealed that Csa-miR160 and their target gene CsARFs were differentially expressed in differential cucumber tissues and developmental stages. Csa-miR160d was only expressed in the expanded cucumber fruit. CsARF5, CsARF11 and CsARF13 exhibited the lower expression in the expanded fruit than those in the ovary, while, CsARF14 showed the reverse trend. Our results suggested that Csa-miR160d might play a crucial role in cucumber fruit expansion by negatively targeting CsARF5, CsARF11 and CsARF13. This is the first genome-wide analysis of miR160 in cucumber. These findings provide useful information and resources for further studying the role of miR160 and ARF in cucumber fruit expansion.

Identification of miRNAs and Their Target Genes Involved in Cucumber Fruit Expansion Using Small RNA and Degradome Sequencing.

Fruit expansion is an essential and very complex biological process. Regulatory roles of microRNAs (miRNAs) and miRNA-mRNA modules in the cucumber fruit expansion are not yet to be investigated. In this work, 1253 known and 1269 novel miRNAs were identified from nine cucumber fruit small RNA (sRNA) libraries through high-throughput sequencing. A total of 105 highly differentially expressed miRNAs were recognized in the fruit on five days post anthesis with pollination (EXP_5d) sRNA library. Further, expression patterns of 11 differentially expressed miRNAs were validated by quantitative real-time PCR (qRT-PCR). The expression patterns were similar to sRNAs sequencing data. Transcripts of 1155 sequences were predicted as target genes of differentially expressed miRNAs by degradome sequencing. Gene Ontology (GO) enrichment showed that these target genes were involved in 24 biological processes, 15 cell components and nine molecular functions. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis demonstrated that these target genes were significantly enriched in 19 pathways and the enriched KEGG pathways were associated with environmental adaptation, signal transduction and translation. Based on the functional prediction of miRNAs and target genes, our findings suggest that miRNAs have a potential regulatory role in cucumber fruit expansion by targeting their target genes, which provide important data for understanding the miRNA-mediated regulatory networks controlling fruit expansion in cucumber. Specific miRNAs could be selected for further functional research and molecular breeding in cucumber.