Dataset Information

MiR-RACE, a new efficient approach to determine the precise sequences of computationally identified trifoliate orange (Poncirus trifoliata) microRNAs.

ABSTRACT: BACKGROUND: Among the hundreds of genes encoding miRNAs in plants reported, much more were predicted by numerous computational methods. However, unlike protein-coding genes defined by start and stop codons, the ends of miRNA molecules do not have characteristics that can be used to define the mature miRNAs exactly, which made computational miRNA prediction methods often cannot predict the accurate location of the mature miRNA in a precursor with nucleotide-level precision. To our knowledge, there haven't been reports about comprehensive strategies determining the precise sequences, especially two termini, of these miRNAs. METHODS: In this study, we report an efficient method to determine the precise sequences of computationally predicted microRNAs (miRNAs) that combines miRNA-enriched library preparation, two specific 5' and 3' miRNA RACE (miR-RACE) PCR reactions, and sequence-directed cloning, in which the most challenging step is the two specific gene specific primers designed for the two RACE reactions. miRNA-mediated mRNA cleavage by RLM-5' RACE and sequencing were carried out to validate the miRNAs detected. Real-time PCR was used to analyze the expression of each miRNA. RESULTS: The efficiency of this newly developed method was validated using nine trifoliate orange (Poncirus trifoliata) miRNAs predicted computationally. The miRNAs computationally identified were validated by miR-RACE and sequencing. Quantitative analysis showed that they have variable expression. Eight target genes have been experimentally verified by detection of the miRNA-mediated mRNA cleavage in Poncirus trifoliate. CONCLUSION: The efficient and powerful approach developed herein can be successfully used to validate the sequences of miRNAs, especially the termini, which depict the complete miRNA sequence in the computationally predicted precursor.

SUBMITTER: Song C

PROVIDER: S-EPMC2881865 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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MiR-RACE, a new efficient approach to determine the precise sequences of computationally identified trifoliate orange (Poncirus trifoliata) microRNAs.

Song Changnian C Fang Jinggui J Wang Chen C Guo Lei L Nicholas Kibet Korir KK Ma Zhengqiang Z

PloS one 20100607 6

<h4>Background</h4>Among the hundreds of genes encoding miRNAs in plants reported, much more were predicted by numerous computational methods. However, unlike protein-coding genes defined by start and stop codons, the ends of miRNA molecules do not have characteristics that can be used to define the mature miRNAs exactly, which made computational miRNA prediction methods often cannot predict the accurate location of the mature miRNA in a precursor with nucleotide-level precision. To our knowledg ...[more]

PMID: 20539756

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Project description:BackgroundMicroRNAs (miRNAs) play a critical role in post-transcriptional gene regulation and have been shown to control many genes involved in various biological and metabolic processes. There have been extensive studies to discover miRNAs and analyze their functions in model plant species, such as Arabidopsis and rice. Deep sequencing technologies have facilitated identification of species-specific or lowly expressed as well as conserved or highly expressed miRNAs in plants.ResultsIn this research, we used Solexa sequencing to discover new microRNAs in trifoliate orange (Citrus trifoliata) which is an important rootstock of citrus. A total of 13,106,753 reads representing 4,876,395 distinct sequences were obtained from a short RNA library generated from small RNA extracted from C. trifoliata flower and fruit tissues. Based on sequence similarity and hairpin structure prediction, we found that 156,639 reads representing 63 sequences from 42 highly conserved miRNA families, have perfect matches to known miRNAs. We also identified 10 novel miRNA candidates whose precursors were all potentially generated from citrus ESTs. In addition, five miRNA* sequences were also sequenced. These sequences had not been earlier described in other plant species and accumulation of the 10 novel miRNAs were confirmed by qRT-PCR analysis. Potential target genes were predicted for most conserved and novel miRNAs. Moreover, four target genes including one encoding IRX12 copper ion binding/oxidoreductase and three genes encoding NB-LRR disease resistance protein have been experimentally verified by detection of the miRNA-mediated mRNA cleavage in C. trifoliata.ConclusionDeep sequencing of short RNAs from C. trifoliata flowers and fruits identified 10 new potential miRNAs and 42 highly conserved miRNA families, indicating that specific miRNAs exist in C. trifoliata. These results show that regulatory miRNAs exist in agronomically important trifoliate orange and may play an important role in citrus growth, development, and response to disease.

Project description:Trifoliate orange (Poncirus trifoliata (L.) Raf.) is extremely cold hardy after a full acclimation; however the underlying molecular mechanisms underlying this economically valuable trait remain poorly understood. In this study, global transcriptome profiles of trifoliate orange under cold conditions (4 °C) over a time course were generated by high-throughput sequencing.More than 68 million high-quality reads were produced and assembled into a non-redundant data of 77,292 unigenes with an average length of 1112 bp (N50 = 1778 bp). Of these, 23,846 had significant sequence similarity to known genes and these were assigned to 61 gene ontology (GO) categories and 25 clusters of orthologous groups (COG) involved in 128 KEGG pathways. Sequences derived from cold-treated and control plants were mapped to the assembled transcriptome, resulting in the identification of 5549 differentially expressed genes (DEGs). These comprised 600 (462 up-regulated, 138 down-regulated), 2346 (1631 up-regulated, 715 down-regulated), and 5177 (2702 up-regulated, 2475 down-regulated) genes from the cold-treated samples at 6, 24 and 72 h, respectively. The accuracy of the RNA-seq derived transcript expression data was validated by analyzing the expression patterns of 17 DEGs by qPCR. Plant hormone signal transduction, plant-pathogen interaction, and secondary metabolism were the most significantly enriched GO categories amongst in the DEGs. A total of 60 transcription factors were shown to be cold responsive. In addition, a number of genes involved in the catabolism and signaling of hormones, such as abscisic acid, ethylene and gibberellin, were affected by the cold stress. Meanwhile, levels of putrescine progressively increased under cold, which was consistent with up-regulation of an arginine decarboxylase gene.This dataset provides valuable information regarding the trifoliate orange transcriptome changes in response to cold stress and may help guide future identification and functional analysis of genes that are importnatn for enhancing cold hardiness.

Project description:Long non-coding RNAs (lncRNAs) play important roles in plant growth and stress responses. As a dominant abiotic stress factor in soil, boron (B) deficiency stress has impacted the growth and development of citrus in the red soil region of southern China. In the present work, we performed a genome-wide identification and characterization of lncRNAs in response to B deficiency stress in the leaves of trifoliate orange (Poncirus trifoliata), an important rootstock of citrus. A total of 2101 unique lncRNAs and 24,534 mRNAs were predicted. Quantitative real-time polymerase chain reaction (qRT-PCR) experiments were performed for a total of 16 random mRNAs and lncRNAs to validate their existence and expression patterns. Expression profiling of the leaves of trifoliate orange under B deficiency stress identified 729 up-regulated and 721 down-regulated lncRNAs, and 8419 up-regulated and 8395 down-regulated mRNAs. Further analysis showed that a total of 84 differentially expressed lncRNAs (DELs) were up-regulated and 31 were down-regulated, where the number of up-regulated DELs was 2.71-fold that of down-regulated. A similar trend was also observed in differentially expressed mRNAs (DEMs, 4.21-fold). Functional annotation of these DEMs was performed using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, and the results demonstrated an enrichment of the categories of the biosynthesis of secondary metabolites (including phenylpropanoid biosynthesis/lignin biosynthesis), plant hormone signal transduction and the calcium signaling pathway. LncRNA target gene enrichment identified several target genes that were involved in plant hormones, and the expression of lncRNAs and their target genes was significantly influenced. Therefore, our results suggest that lncRNAs can regulate the metabolism and signal transduction of plant hormones, which play an important role in the responses of citrus plants to B deficiency stress. Co-expression network analysis indicated that 468 significantly differentially expressed genes may be potential targets of 90 lncRNAs, and a total of 838 matched lncRNA-mRNA pairs were identified. In summary, our data provides a rich resource of candidate lncRNAs and mRNAs, as well as their related pathways, thereby improving our understanding of the role of lncRNAs in response to B deficiency stress, and in symptom formation caused by B deficiency in the leaves of trifoliate orange.

Dataset Information

MiR-RACE, a new efficient approach to determine the precise sequences of computationally identified trifoliate orange (Poncirus trifoliata) microRNAs.

Publications

MiR-RACE, a new efficient approach to determine the precise sequences of computationally identified trifoliate orange (Poncirus trifoliata) microRNAs.

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