Unknown,Transcriptomics,Genomics,Proteomics

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Identification of microRNAs in Caragana intermedia by high-throughput sequencing and expression analysis of 12 microRNAs and their targets under salt stress

ABSTRACT: MicroRNAs (miRNAs) are endogenous small RNAs that play important roles in various biological and metabolic processes in plants. Caragana intermedia is an important ecological and economic tree species that is prominent in the desert environment of west and northwest China. To date, no investigation into C. intermedia miRNAs has been reported. In this study, high-throughput sequencing of small RNAs was performed to identify both conserved and novel miRNAs, and also their target mRNA genes in C. intermedia. Based on sequence similarity and hairpin structure prediction, 132 putative conserved miRNAs (12 of which were confirmed to form hairpin precursors) belonging to 31 known miRNA families were identified. Ten novel miRNAs (including the miRNA* sequences of three novel miRNAs) were also discovered. The expression of 12 miRNAs was validated in different tissues, and the 12 miRNAs were further assessed by qRT-PCR after salt treatment. Furthermore, 36 potential target genes of 17 known miRNA families and two potential target genes of one novel miRNA were predicted, and some target genes were also assessed by qRT-PCR after salt treatment. Our study provides a basic catalog of miRNAs and their targets, which will promote further understanding of the important roles of miRNAs in C. intermedia and in other species of the leguminous genus, Caragana. Examination of small RNA expression profiling in three-weeks-old seedings of Caragana intermedia.

ORGANISM(S): Caragana korshinskii

SUBMITTER: Zhu Jianfeng

PROVIDER: E-GEOD-44819 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background: The halophyte Mesembryanthemum crystallinum (ice plant) is a model for studying salt tolerance. The morphology, physiology, metabolism, and gene expression of ice plant have been studied for over 40 years. Although the complete genome sequence has not been revealed, large-scale analyses of gene expression profiling have drawn an outline of salt tolerance in ice plant. Despite ample information in the transcriptome, miRNA information has not been documented. Results: We examined responses to a sudden increase in salinity in ice plant seedlings. Using a fluorescent dye to detect Na+, we found that ice plant roots respond to an increased flux of Na+ by either secreting or storing Na+ in specialized cells. High-throughput sequencing was used to identify small RNA profiles in three-day-old seedlings treated with or without 200 mM NaCl. Totally 132 conserved miRNAs belonging to 22 families were found. The hairpin precursor of 19 conserved mcr-miRNAs and 12 novel mcr-miRNAs were identified. Target genes are involved in a broad range of biological processes: transcription factors that regulate growth and development, enzymes that catalyze miRNA biogenesis for the most conserved mcr-miRNA, and proteins that are involved in ion homeostasis and drought-stress responses for some novel mcr-miRNAs. After 6 h of salt stress, the expressions of most mcr-miRNAs were down-regulated, whereas the expressions of their corresponding target genes were up-regulated. Analyses of the functions of target genes revealed that cellular processes, including growth and development, metabolism, and ion transport activity were up-regulated in roots under salt stress. Conclusions: Analyses of small RNA profile of ice plant seedlings identified many conserved miRNA families and several novel miRNAs. The expression of ten conserved miRNAs and three novel miRNAs were reciprocally correlated to predicted targets hourly after salt stress. Based on the expression pattern of miRNA and target genes in combination with the observation of Na+ distribution, we suggest that ice plant roots respond effectively to increased salinity by using Na+ as an osmoticum for cell expansion and guard cell opening. Excessive Na+ could either be secreted through root epidermis or stored in specialized leaf epidermal cells. These responses are partially regulated at the miRNA-mediated post-transcriptional level.

Project description:Backgroud: microRNA (miRNA) is implicated in plant development processes, playing pivotal roles in plant adaptation to environmental stresses. Salicornia europaea, a salt mash euhalophyte, is a good model plant to study salt adaptation mechanisms. It is also attractive in being vegetables, forage and oilseed that can be used for saline land reclamation and biofuel precursor production on marginal lands. However, none of the miRNAs from S. europaea have been identified so far. Results: Deep sequencing was performed to investigate small RNA transcriptome of S. europaea. Two hundred and twelve conserved miRNAs comprising 51 families and 31 novel miRNAs (including 7 miRNA star sequences) belonging to 30 families were identified. Interestingly, about half (13 out of 31) of the novel miRNAs were only detected in salt-treated samples. The expression of 43 conserved and 13 novel miRNAs changed significantly in response to salinity. In addition, 53 conserved miRNAs and 13 novel miRNAs were differentially expressed between shoots and roots. Furthermore, a total of 306 and 195 S. europaea unigenes were predicted to be targets of 41 conserved and 29 novel miRNA families, respectively. These targets encode a wide range of proteins, and genes involved in transcription regulation constitute the largest category. Four of them, which encode laccase, F-box family protein, SAC3/GANP family protein, and nadph-cytochrome P450 oxydoreductase, were validated using 5'-RACE. Conclusions: Our results indicate specific miRNAs are tightly regulated by salinity in shoots and/or roots of S. europaea, which play important roles in salt adaptation of this euhalophyte. The S. europaea salt-responsive miRNAs and miRNAs that target transcription factors, nucleotide binding site-leucine-rich repeat proteins and enzymes involved in lignin biosynthesis as well as carbon and nitrogen metabolism may be applied in genetic engineering of crops with higher stress tolerance, and genetic modification of biofuel crops with higher biomass and regulatable lignin biosynthesis.

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Identification of microRNAs in Caragana intermedia by high-throughput sequencing and expression analysis of 12 microRNAs and their targets under salt stress

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