Abstract
Protoplast isolation is a stress-inducing process, during which a variety of physiological and molecular alterations take place. Such stress response affects the expression of totipotency of cultured protoplasts. MicroRNAs (miRNAs) play important roles in plant growth, development and stress responses. However, the underlying mechanism of miRNAs involved in the protoplast totipotency remains unclear. In this study, high-throughput sequencing technology was used to sequence two populations of small RNA from calli and callus-derived protoplasts in Citrus reticulata Blanco. A total of 67 known miRNAs from 35 families and 277 novel miRNAs were identified. Among these miRNAs, 18 known miRNAs and 64 novel miRNAs were identified by differentially expressed miRNAs (DEMs) analysis. The expression patterns of the eight DEMs were verified by qRT-PCR. Target prediction showed most targets of the miRNAs were transcription factors. The expression levels of half targets showed a negative correlation to those of the miRNAs. Furthermore, the physiological analysis showed high levels of antioxidant activities in isolated protoplasts. In short, our results indicated that miRNAs may play important roles in protoplast-isolation response.
Highlights
IntroductionMicroRNAs (miRNAs) are a class of endogenous, small non-coding RNAs with length of 20– 24 nucleotides
MicroRNAs are a class of endogenous, small non-coding RNAs with length of 20– 24 nucleotides
17,975,734 and 15,828,591 total reads were obtained from callus library and protoplast library, respectively. These total reads were cleaned by removing adaptors, contaminants, and low quality tags, generating 17,899,040 (99.74%) and 15,663,034 (99.22%) clean reads including miRNA, rRNA, snRNA, snoRNA, tRNA, degraded fragments of mRNA introns or exons and several other unannotated reads (Table 1)
Summary
MicroRNAs (miRNAs) are a class of endogenous, small non-coding RNAs with length of 20– 24 nucleotides. Since the first miRNA, lin-4, was discovered in Caenorhabditis elegans [4], lots of miRNAs have been identified in animals, plants, and viruses. According to the miRNAs database (miRBase 21, July 2014), 8,496 mature miRNAs were found in 73 plant species including 53 dicotyledons, 12 monocotyledons, 4 conifers, Chlamydomonas reinhardtii, Physcomitrella patens, Selaginella moellendorffii, and Amborella trichopoda. Increasing evidence suggest that plant miRNAs play important roles in all almost biological and metabolic processes, including leaf development [5], shoot branching [6], root growth [7,8,9], control of flowering time and floral organ identity.
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