Identification And Different Expression Of Dendrobium Officinale MiRNAs

MicroRNAs (miRNAs) are endogenous~22nt noncoding RNAs that haveimportant functions in the regulation of gene expression at post-transcriptional levelby pairing the mRNA for cleavage or translational inhibition. MiRNAs typically actwithin the RNA-induced silencing complex (RISC) and down-regulate the expressionof the complementary target mRNA in the untranslated regions by endoribonucleaseargonaute proteins. Previous studies have revealed that miRNAs participate in certainphysiological activities, including growth regulation, proliferation, differentiation, anddeath.To date, a large number of miRNAs have been identified from numerous plants,including Oryza sativa, Arabidopsis thaliana and Zea mays. Plant miRNA sequencesare available in the Sanger miRNA database. However, no Dendrobium officinalemiRNA is available in miRBase. Therefore, the identification of miRNA from D.officinale is important to investigate their biological functions further.D. officinale is a monocotyledonous perennial herbaceous plant that belongs toOrchidaceae family. This plant is extensively used in China, and has been animportant and invaluable herb in traditional Chinese medicine. D. officinale hasnumerous pharmacological activities, including anti-oxidation, immunomodulation,anti-fatigue, digestion promotion, anti-hypertension, and antitumor activities. In China,the number of D. officinale is gradually declining because of the low rate ofpropagation in natural conditions and overexploitation. Nearly all D. officinalematerials are collected from artificial cultivation by transplanting tissue-cultureseedlings into fields. In natural or artificial culture condition, the growth rate of D.officinale is quite low and plant endophyte is indispensable for D. officinale growth.In micropropagated condition, the culture medium supplemented with auxin which isindispensable for plant growth is used for micropropagated cultivation, indicating thatauxin may be the key factor for the growth of D. officinale in micropropagatedcondition. To determine whether different cultural conditions affect the expression ofD. officinale miRNA between conventional and micropropagated D. officinale, we reported the differential expression profiles of miRNA by microarray in conventionaland micropropagated D. officinale plants. A total of120miRNA from37miRNAfamilies were identified in both conventional and micropropagated D. officinale plants.In addition, we investigated the homology between the identified miRNA and theknown miRNA of other species. O. sativa showed the most homology with D.officinale, implying that the existence of conserved miRNA was stable amongdifferent plant species.Based on the signals of microarray, we selected45miRNA sequences from sixmiRNA families that were significantly differentially expressed. The results showedthat miR156, miR164, miR171, miR529and miR827expressed at very high levelswhile miR167expressed at very low levels in micropropagated D. officinale,indicating that the expression of these miRNAs could be up/down-regulated bymicropropagated culture condition. Based on the results of computational predictionand GO analysis, most of the differentially expressed miRNA were mainly involvedin developmental regulation, nucleic acid binding, and stimulus response. We selected6representative miRNAs for Quantitative real-time RT–PCR (qRT–PCR) validation.The results showed that miR156g, miR529b and miR827a expressed at very highlevels while miR159d, miR167h and miR528expressed at very low levels inmicropropagated D. officinale. In general, the results of qRT–PCR showedreproducible correlation with the microarray data. Our research offers a foundation forthe further investigation of the functional regulatory roles of miRNAs and their targetgenes in D. officinale.