Identification Of MiRNAs From The Salivary Gland And Functional Study Of MiR-306-5p In Plutella Xylostella (L.)

microRNAs (miRNA) are a class of endogenous non-coding RNAs that, take part in many processes associated with development and immunity by modulating gene expression at the post-transcriptional level. The diamondback moth, Plutella xylostella (L.), is an important insect pest of cruciferous plants. It is still unclear whether miRNAs are connecting the interactions between P. xylostella and its host plants. In this study, we sequenced a small RNA (sRNA) library from the salivary gland of P. xylostella. Based on bioinformatic approachs, a number of miRNAs and their potential gene targets were predicted. The miRNAs with potential target genes involving in glucosinolate biosynthesis in Arabidopsis thaliana were further screened, and were used for target genes identification in P. xylostella.The length distribution of salivary gland sRNA of P. xylostella ranged from 22 to 26 nt, which included 774 conserved and 545 novel miRNAs. Based on this, the miRNA expression profiles were built. A total of 47,822 genes are predicted to be the targets of conserved and novel miRNAs. GO analysis showed that their functions were mainly focused on catalytic activity and binding, and are also involved in cellular and metabolic processes, stimulus response and signaling pathways, etc. KEGG analysis indicated that the pathways of metabolism, protein digestion, absorption and secretion were significantly enriched.Five miRNAs of P. xylostella that were predicted to target genes involved in glucosinolate biosynthesis in A. thaliana were selected for T-A cloning, Sanger sequencing and sequence alignment. One miRNA, miR-306a-5p, was comfirmed to be conserved among the insects and have homologous counterpart in crucifers. Based on the open reading frame and untranslated regions of genes in P. xylostella, potential target genes of the miR-306a-5p were predicted, eight of which were further studied. The expression pattern analysis at different developmental stages showed that miR-306a-5p is expressed in a reverse manner compared to its potential target genes Px013190, Px010939, Px001479, Px000505 and Px014036. The miR-306a-5p had higher expression levels both in 3rd instar larva and pupa, while that of its predicted target genes were on the opposite. Cell transfection of P. xylostella with miR-306a-5p mimic and inhibitor found that the potential target genes Px013199, Px001479, Px000505 and Px014036 were suppressed in the mimic treatment, however, no obvious up-regulation of the potential target genes could be observed in the inhibitor treatment. Transcriptome sequencing and analysis for the cells subjected to the mimic and inhibitor treatments showed that differentially expressed genes Px003058 and Px006077 had reverse expression patterns between the two treatments, which were not in the list of potential target genes predicted previously.The study promises to explore the roles of miRNA involved with insect-plant interaction and adaptation of P. xylostella to its host plants, and provides the theoretical basis of miRNA-based control of P. xylostella.