Genome-wide identification of drought-responsive microRNAs and their target genes in Chinese jujube by deep sequencing

Abstract

Background: MicroRNAs (miRNAs) are about 21 snucleotide (nt) long, non-coding RNAs that play an important role in plant abiotic stress responses. Chinese jujube is a native fruit tree in China, which is also an admittedly drought-resistant plant. But the drought-related miRNAs have little been reported in jujube.

Objective: To identify possibly drought-responsive microRNAs and their target genes in Chinese Jujube.

Methods: Twelve small RNA libraries were constructed from two jujube genotypes both drought treated and control samples with three replicates to identify known and novel miRNAs in Chinese Jujube, DESeq2 was used to identify expression pattern of miRNAs between drought treatment and control samples, TargetFinder program was used to predict potential target genes of conserved and novel miRNAs, RT-qPCR were used to analysis the expression levels of drought-related miRNAs and their potential targets. The RNA ligase-mediated RLM-5' RACE experiments were performed to validate predicted target genes of drought-related miRNAs.

Results: 43 known miRNAs and 431 novel miRNAs were identified in Chinese jujube. Expression analysis showed that 28 miRNAs were differential expressed under drought stress in jujube variety "Dongzao", including 21 up-regulated miRNAs and 7 down-regulated miRNAs, 61 miRNAs were differential expressed under drought stress in Chinese jujube variety "Zanhuangdazao", including 23 up-regulated miRNAs and 37 down-regulated miRNAs. Depend on miRNAs target prediction, functional annotation and expression analysis, we identified 9 drought-related miRNAs, and 7 target genes of 6 miRNAs were confirmed using the modified 5'-RACE method. Also, RT-qPCR analyses revealed that relative expression of those miRNAs and their targets have negative tendency.

Conclusion: We identified 6 drought-related miRNAs by high-throughout sequencing and target gene annotation from Chinese jujube, and targets of those miRNAs were confirmed by the modified 5'-RACE method. These findings provide molecular evidence for enhancing drought tolerance in Chinese jujube and other plants.

Keywords: Deep sequencing; Drought stress; Jujube; MircoRNA.