Identification and Differential Expression of MicroRNAs during Cold Acclimation of the Tea Plant (Camellia sinensis) by Deep Sequencing
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ABSTRACT: In this study, we performed deep sequencing and bioinformatics analyses of tea plant leaves to identify and characterize known and novel miRNAs. A total of 26,876,261 raw reads were produced from 2 libraries. We detected 422 known miRNAs belonging to 125 families, and 68 putative novel miRNAs.
Project description:Solexa sequencing technology was used to perform high throughput sequencing of the small RNA library from the cold treatment of tea leaves. Subsequently, aligning these sequencing date with plant known miRNAs, we characterized 112 C. sinensis conserved miRNAs. In addition, 215 potential candidate miRNAs were found; among them, 131 candidates with star sequence were chosen as novel miRNAs. There are both congruously and differently regulated miRNAs, and line-specific miRNAs were identified by microarray-based hybridization in response to cold stress. The miRNA chip included 3228 miRNA probes corresponding to miRNA transcripts listed in Sanger miRBase release 19.0 and 283 novel miRNAs probes founding in tea plant. In the study presented here, two tea plant cultivars, ‘Yingshuang’ (YS, a cold-tolerant tea plant cultivar) and ‘Baiye 1’ (BY, a cold-sensitive tea plant cultivar), were kept at 4°C for 4,12, 24 h, respectively, and 28°C for as control. These samples were used to acquire expression profiles of a total of 3,511 unique genes, leading to the successful construction of supervised
Project description:Solexa sequencing technology was used to perform high throughput sequencing of the small RNA library from the cold treatment of tea leaves. Subsequently, aligning these sequencing date with plant known miRNAs, we characterized 112 C. sinensis conserved miRNAs. In addition, 215 potential candidate miRNAs were found; among them, 131 candidates with star sequence were chosen as novel miRNAs. There are both congruously and differently regulated miRNAs, and line-specific miRNAs were identified by microarray-based hybridization in response to cold stress. The miRNA chip included 3228 miRNA probes corresponding to miRNA transcripts listed in Sanger miRBase release 19.0 and 283 novel miRNAs probes founding in tea plant.
Project description:In this study, it is noticeable that 32 tea-specific miRNAs were confirmed on the base of genome survey, using deep sequencing and microarray hybridization, and many miRNAs might associate with secondary metabolites synthesis. Leaves, buds and roots were collected
Project description:Tea (Camellia sinensis (L.) O. Kuntze) is an important non-alcoholic commercial beverage crop. Tea tree is a perennial plant, and winter dormancy is its part of biological adaptation to environmental changes. We recently discovered a novel tea tree cultivar that can generate tender shoots in winter, but the regulatory mechanism of this ever-growing tender shoot development in winter is not clear. In this study, we conducted a proteomic analysis for identification of key genes and proteins differentially expressed between the winter and spring tender shoots, to explore the putative regulatory mechanisms and physiological basis of its ever-growing character during winter.
Project description:Anthracnose disease is caused by Colletotrichum gloeosporioides, and is common in leaves of the tea plant Camellia sinensis. MicroRNAs (miRNAs) have been known as key modulators of gene expression in defense responses; however, the role of miRNAs in tea plant during defensive responses to C. gloeosporioides remains unexplored. Six miRNA sequencing data sets and two degradome data sets were generated from C. gloeosporioides-inoculated and control tea leaves. A total of 485 conserved and 761 novel miRNAs were identified. Of those, 239 known and 369 novel miRNAs exhibited significantly differential expression under C. gloeosporioides stress. 1134 and 596 mRNAs were identified as targets of 389 and 299 novel and conserved miRNAs by degradome analysis, respectively. The expression levels of twelve miRNAs and their targets were validated by quantitative real-time PCR. The predicted targets of five interesting miRNAs were further validated through 5'RLM-RACE. Furthermore, Gene Ontology and metabolism pathway analysis revealed that most of the target genes were involved in translation, carbohydrate metabolism and signal transduction pathways. This study enriches the resources of defense-responsive miRNAs and their targets in C. sinensis, and thus, provides novel insights into the miRNA-mediated regulatory mechanisms underlying immunity responses to biotic stress in tea plant.
Project description:In this study, it is noticeable that 32 tea-specific miRNAs were confirmed on the base of genome survey, using deep sequencing and microarray hybridization, and many miRNAs might associate with secondary metabolites synthesis.
Project description:We discovered 31 up-regulated miRNAs and 43 down-regulated miRNAs in ‘Yingshuang’, and 46 up-regulated miRNA and 45 down-regulated miRNAs in ‘Baiye 1’ in response to cold stress, respectively. A total of 763 related target genes were detected by degradome sequencing. The RLM-5’RACE procedure was successfully used to map the cleavage sites in six target genes of C. sinensis. These findings reveal important information about the regulatory mechanism of miRNAs in C. sinensis, and promote the understanding of miRNA functions during the cold response. The miRNA genotype-specific expression model might explain the distinct cold sensitivities between tea lines.
Project description:BACKGROUND: Tea is the most popular non-alcoholic health beverage in the world. The tea plant (Camellia sinensis (L.) O. Kuntze) needs to undergo a cold acclimation process to enhance its freezing tolerance in winter. Changes that occur at the molecular level in response to low temperatures are poorly understood in tea plants. To elucidate the molecular mechanisms of cold acclimation, we employed RNA-Seq and digital gene expression (DGE) technologies to the study of genome-wide expression profiles during cold acclimation in tea plants. RESULTS: Using the Illumina sequencing platform, we obtained approximately 57.35 million RNA-Seq reads. These reads were assembled into 216,831 transcripts, with an average length of 356 bp and an N50 of 529 bp. In total, 1,770 differentially expressed transcripts were identified, of which 1,168 were up-regulated and 602 down-regulated. These include a group of cold sensor or signal transduction genes, cold-responsive transcription factor genes, plasma membrane stabilization related genes, osmosensing-responsive genes, and detoxification enzyme genes. DGE and quantitative RT-PCR analysis further confirmed the results from RNA-Seq analysis. Pathway analysis indicated that the "carbohydrate metabolism pathway" and the "calcium signaling pathway" might play a vital role in tea plants' responses to cold stress. CONCLUSIONS: Our study presents a global survey of transcriptome profiles of tea plants in response to low, non-freezing temperatures and yields insights into the molecular mechanisms of tea plants during the cold acclimation process. It could also serve as a valuable resource for relevant research on cold-tolerance and help to explore the cold-related genes in improving the understanding of low-temperature tolerance and plant-environment interactions.
Project description:Recently, intensive global climate change has become a major factor impacting plant survival during the winter. Freezing cold temperatures during the winter and abnormal temperature fluctuations during the winter and early spring are the most harmful ambient factors threatening tea plant winter survival and currently cause marked economic losses in tea production. In this study, by simulating natural climate change, we established cold acclimation (CA) and rapid cold stress (after CA) conditions to comprehensively investigate the transcriptome changes involved in CA and rapid cold stress. Electrolyte leakage (EL) rate and expression profile clustering analyses confirmed that the experimental design was valid. Comparative transcription analysis identified many differentially expressed genes (DEGs) involved in both processes. Time course and pathway enrichment analyses further revealed the physiological changes that occur during the initial period of CA and the cell wall changes that occur throughout the entire CA process; these changes play crucial roles in increasing freezing tolerance during this process. Compared with CA, different cold response mechanisms were rapidly activated under cold stress; however, the subsequent accumulation of reactive oxygen species, which affect multiple aspects, caused by freezing cold could be the harshest factor impairing tea leaves. Moreover, we investigated 60 DEGs shared by both processes and highlighted the importance of KCSs, HXXXD-type acyl-transferase family proteins, NAC080, SWEETs and ENOs in the responses to various cold conditions. These results greatly improve our knowledge of cold response mechanisms in tea plants and provide meaningful information for functional studies investigating cold tolerance-related genes.