In silico identification of miRNAs and their target genes and analysis of gene co-expression network in saffron (Crocus sativus L. ) stigma

Abstract

As an aromatic and colorful plant of substantive taste, saffron (Crocus sativus L.) owes such properties of matter to growing class of the secondary metabolites derived from the carotenoids, apocarotenoids. Regarding the critical role of microRNAs in secondary metabolic synthesis and the limited number of identified miRNAs in C. sativus, on the other hand, one may see the point how the characterization of miRNAs along with the corresponding target genes in C. sativus might expand our perspectives on the roles of miRNAs in carotenoid/apocarotenoid biosynthetic pathway. A computational analysis was used to identify miRNAs and their targets using EST (Expressed Sequence Tag) library from mature saffron stigmas. Then, a gene co- expression network was constructed to identify genes which are potentially involved in carotenoid/apocarotenoid biosynthetic pathways. EST analysis led to the identification of two putative miRNAs (miR414 and miR837-5p) along with the corresponding stem- looped precursors. To our knowledge, this is the first report on miR414 and miR837-5p in C. sativus. Co-expression network analysis indicated that miR414 and miR837-5p may play roles in C. sativus metabolic pathways and led to identification of candidate genes including six transcription factors and one protein kinase probably involved in carotenoid/apocarotenoid biosynthetic pathway. Presence of transcription factors, miRNAs and protein kinase in the network indicated multiple layers of regulation in saffron stigma. The candidate genes from this study may help unraveling regulatory networks underlying the carotenoid/apocarotenoid biosynthesis in saffron and designing metabolic engineering for enhanced secondary metabolites.

Keywords: Co-expression network; Crocus sativus; EST sequences analysis.

Figure 1

Predicted secondary structure of in silico identified precursor miRNAs in C. sativus L. The mature miRNAs sequences are underlined and highlighted in red

Figure 2

A graphical view of co-expression network of potential target genes. Network was constructed using GeneMANIA based on automatically selected weighting method. Network includes predicted miRNAs, miRNA-target genes and co-expressed genes. Relations between miRNAs and target genes are shown as black edges, whereas relations between target genes and co-expressed genes are depicted by gray edges. Lycopene beta/epsilon cyclase protein (AT2G32640) was directly or indirectly connected to almost all of the target genes of predicted miR414 and miR837-5P. The co-expression network also unraveled the relationship between beta carbonic anhydrase 5 (a target of miR414) and alpha-1,2- glucosyltransferase (T1E22_170). Genes involved in carotenoid/apocarotenoid biosynthesis (AT2G32640 and T1E22_170) are highlighted with a circle