Identification And Expression Of MicroRNAs And Their Targets In Caragana Intermedia

Caragana intermedia belongs to the Fabaceae family, and is a native desert shrub withstrong drought-resistance, sand-fixing capacity and high forage value. It is widely distributedin the desert land of west and northwest China, and is a very important ecological andeconomic species. Plant MicroRNA (miRNA) is widely distributed in the plant genome and isa class of short, single-stranded, endogenous, non-coding RNAs, approximately19-25nucleotides (nt) long. Accumulating data demonstrate that miRNAs play major roles in manybiological and metabolic processes, including developmental timing, tissue identity, andresponses to biotic and abiotic stress. It is very significance to carry out identification andexpression of miRNAs and their target genes in important ecological and economic species, soas to achieve effective control and use of important genes from miRNAs to explain theimportant phenomenon of life. More important is to achieve effective regulation of importantgenes with the purpose of using them. In this study, high-throughput sequencing of smallRNAs and analysis of transcriptome data were performed to identify both conserved and novelmiRNAs, and also their target mRNA genes in C. intermedia. Some target mRNAs werevalidated by5′RACE, and miRNAs was validated by qRT-PCR. Using qRT-PCR,10candidatereference genes were analyzed in C. intermedia subjected to different organs in differentdevelopmental stages and different abiotic (osmotic, salt, cold and heat) stresses, theexpression stability of these genes was assessed using geNorm, NormFinder and BestKeeperalgorithms. Using qRT-PCR, expression analyses of miRNAs and miRNA target mRNAsduring different organs in different developmental stages and NaCl stress conditions in C.intermedia. The main results are as follows:(1) Sequencing results generated24,543,122raw reads form seedling,20,179,252rawreads form vegetative organs of mature period and20,209,482raw reads form reproductionorgans of mature period. After removing the adapter sequences, low quality tags and readsshorter than18nt, we respectively obtained24,147,728,19,748,030and19,762,567clean reads from the3small RNA libraries. Among the clean reads,4,956,618(20.53%),1,181,916(5.98%) and1,623,033(8.21%) clean reads were respectively matched to the known plantmiRNA.18,477,635(76.52%),17,446,062(88.34%) and17,005,152(86.05%) were found tobe unannotated small RNAs, the high number of small RNAs that were not annotatedsuggested that there may be many novel miRNAs in C. intermedia.(2) Based on sequence similarity and hairpin structure prediction160putative conservedmiRNAs belonging to40known miRNA families were identified.16novel miRNAs were alsodiscovered. The expression detection of14conserved and2novel miRNAs were validated byqRT-PCR, which demonstrated that next-generation high-throughput sequencing combinedwith bioinformatic analysis is a powerful tool for miRNA discovery.(3) A total of45targets for the conserved and novel miRNAs were predicted andannotated, which involved in all kinds of biological processes including developmental timing,tissue identity, responses to biotic and abiotic stress and so on. We validated5of them by5′RACE and noticed that the major cleavage sites in the miRNA targets were not in the centerof the binding sites.(4) Using qRT-PCR,10candidate reference genes were analyzed in C. intermediasubjected to different organs in different developmental stages and different abiotic (osmotic,salt, cold and heat) stresses. The expression stability of these genes was assessed using geNorm,NormFinder and BestKeeper algorithms. UNK2, SAND, PP2A and EF-1a would be appropriatefor normalizing gene expression data for different organs in different developmental stages.UNK2and SAND would be appropriate for normalizing gene expression data for salt-treatedroots and leaves. UNK1, UNK2and PP2A would be appropriate for PEG-treated (osmotic)roots, whereas the combination of TIP41and PP2A was the most suitable for PEG-treatedleaves. SAND, PP2A and TIP41exhibited the most stable expression in heat-treated leaves. Incold-treated leaves, SAND and EF-1α were the most stably expressed. This study will benefitfuture studies on gene expression in C. intermedia.(5) The expression patterns of the11miRNAs and their targets during different organs indifferent developmental stages were assessed by qRT-PCR, the results showed that the expression patterns of many miRNAs are temporal and spatial specificity.10miRNAs arehighly expressed in vegetative organs of mature period; one miRNA (cin-miR157a) is highlyexpressed in juvenile stage. and three targets showed an approximately negative correlationwith their corresponding miRNAs during different developmental stages.The results also showthat the C. intermedia miRNAs and its target genes existence relation to growth and decline indifferent developmental stages and different organs. High expression of miRNA and its targetgene expression is often low; when the miRNA expression is low, and its target geneexpression is often high. This result indicates that miRNA may play important roles during thegrowth and development of C. intermedia.(6) The expression patterns of the11miRNAs and their targets in leaves were assessed byqRT-PCR after salt treatment. The expression levels of7miRNAs were upregulated while onemiRNA (cin-miR398a) was downregulated after salt-treatment, and four targets showed anapproximately negative correlation with their corresponding miRNAs under salt treatment.This result indicates that the target mRNAs (growth and development-related genes) arerepressed by the high expression of their corresponding miRNAs or the target mRNAs (stresstolerance-related genes) are relieved by the low expression of their corresponding miRNAs inC. intermedia during adaptation to abiotic stress.In conclusion,160conserved miRNAs were identified, and16novel miRNAs were alsodiscovered,45targets for the conserved and novel miRNAs were predicted and annotated;Superior reference genes were selected and which will benefit future studies on geneexpression in C. intermedia; The expression patterns of the11miRNAs and their targets indifferent developmental stages and salt stress were assessed by qRT-PCR, the result indicatesthat miRNA may play important roles during the growth and development and abiotic stress ofC. intermedia. This study will benefit future studies on miRNA research and improve breedingof C. intermedia.