Identification of copper (Cu) stress-responsive grapevine microRNAs and their target genes by high-throughput sequencing
ABSTRACT: Background: MicroRNAs (miRNAs) are a class of single-stranded non-coding small RNAs (sRNAs) that are 20-24 nucleotides (nt) in length. Extensive studies have indicated that miRNAs play important roles in plant growth, development, and stress responses. With more copper (Cu) and copper-containing compounds used as bactericides and fungicides in viticulture, Cu stress has become one of the serious environmental problems that affect plant growth and development. In order to uncover the hidden response mechanisms of Cu stress, many Cu-responsive miRNAs have been detected in several plant species. However, there have been few reports about the grapevine miRNAs in response to Cu. Results: Here, two small RNA libraries were constructed from Cu-treated and water-treated (control) leaves of 'Summer Black' grapevine. Following high-throughput sequencing and filtering, 158 known vvi-miRNAs and 98 novel vvi-miRNAs were identified in the two libraries. Among these, 24 could only be detected in the treatment, and 63 were only detected in the control. Additionally, 100 known vvi-miRNAs were found to be clearly responsive to Cu, among which 96 were down-regulated and four were up-regulated; 47 novel vvi-miRNAs were found to be clearly responsive to Cu, among which 35 were down-regulated and 12 were up-regulated. Subsequently, expression patterns of a set of Cu-responsive vvi-miRNAs were validated by quantitative real-time PCR (qRT-PCR). There existed some consistency in expression levels of Cu-responsive vvi-miRNAs between high-throughput sequencing and qRT-PCR assays. In addition, 92 putative targets for 79 known vvi-miRNAs and 51 putative targets for 22 novel vvi-miRNAs were predicted, and most of the targets are involved in multiple biological processes including transcriptional regulation and response to biotic and abiotic stresses. Conclusions: In this study, 147 Cu-responsive vvi-miRNAs were identified using high-throughput sequencing, and their target genes were predicted, which will be helpful to understanding the molecular mechanisms of miRNAs in response to Cu stress. Furthermore, this work can also provide a foundation for further study of the networks of miRNAs involved in grapevine plant growth and breeding some Cu-tolerant grapevine cultivars. Mixed 'Summer Black' grapevine young leaves (2 weeks old), large leaves (5 weeks old), and old leaves (9 week old) in randomly-selected plants from both the Cu-treated and control groups were collected for high-throughput sequencing. Subsequently, we carried out the analysis of Solexa sequencing data, and performed the research of regulatory modes of grapevine miRNAs on their target genes during Cu stress.
Project description:The present study was designed to find out the transcriptional system that is connected with the development of leaves and production of metabolites during photosynthesis. We examine the cellular expression by use the up-to-date RNA sequencing expertise to carry out a complete analysis of the transcriptional profile of V. vinifera leaves at four different developmental stages. Total RNA extracted from 4 samples with three replication for each and compared within all samples.
Project description:The systematic deep sequencing analysis provided a comprehensive understanding of the transcriptome complexity of 2n and 3n Fujian oyster. This information broadens our understanding of the mechanisms of C.angulata polyploidization and contributes to molecular and genetic research by enriching the oyster database. This is the first report on genome-wide transcriptional analysis of adductor muscle of diploid and triploid Fujian oyster and has demonstrated triploid oysters are morphologically almost identical to their diploid counterparts, but have faster growth, due to the reorientation of energetic allocation from gametogenesis to somatic investment. This study provides a foundation for further analysis of the gene expression patterns and signaling pathways which regulate the molecular mechanisms of diploid and triploid oyster. Examination of 3 different samples, including diploid (DF and DM) and triplod(T) oyster.
Project description:MicroRNAs (miRNAs) play a important part 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 and other plants. However, the number of miRNAs discovered in grape is relatively low and little is known about miRNAs responded gibberellin during fruit germination. In this study, a small RNA library from gibberellin grape fruits was sequenced by the high throughput sequencing technology. A total of 16,033,273 reads were obtained. 812,099 total reads representing 1726 unique sRNAs matched to known grape miRNAs. Further analysis confirmed a total of 149 conserved grapevine miRNA (Vv-miRNA) belonging to 27 Vv-miRNA families were validated, and 74 novel potential grapevine-specific miRNAs and 23 corresponding novel miRNAs* were discovered. Twenty-seven (36.5%) of the novel miRNAs exhibited differential QRT-PCR expression profiles in different development gibberellin-treated grapevine berries that could further confirm their existence in grapevine. QRT-PCR analysis on transcript abundance of 27 conserved miRNA family and the new candidate miRNAs revealed that most of them were differentially regulated by the gibberellin, with most conserved miRNA family and 26 miRNAs being specifically induced by gibberellin exposure. All novel sequences had not been earlier described in other plant species. In addition, 117 target genes for 29 novel miRNAs were successfully predicted. Our results indicated that miRNA-mediated gene expression regulation is present in gibberellin-treated grape berries. This study led to the confirmation of 101 known miRNAs and the discovery of 74 novel miRNAs in grapevine. Identification of miRNAs resulted in significant enrichment of the gibberellin of grapevine miRNAs and provided insights into miRNA regulation of genes expressed in grape berries. GSM604831 is the control for the gibberellin-treated sample. The mixture samples of young berries (one week after flowering) large berries (five week after flowering after flowering), and old berries (nine week after flowering) treated with gibberellin, respectively, were generated by deep sequencing, in triplicate, using Illumina 1G Genome Analyzer.
Project description:MicroRNA (miRNA) is a class of functional non-coding small RNA with 19-25 nucleotides in length. Amur grape (Vitis amurensis Rupr.) is an important wild fruit crop with the strongest cold resistance in the Vitis genus and is used as an excellent breeding parent for grapevine, and with growing interest in terms of wine production. To date, there is a relatively large number of grapevine miRNAs (vv-miRNAs) from cultivated grapevine varieties such as Vitis vinifera L. and hybrids of V. vinifera and V. labrusca, but there is no report on miRNAs from Vitis amurensis Rupr, a wild grapevine species. In this study, a small RNA library from Amur grapes was constructed and Solexa technology used to perform deep sequencing of the library followed by subsequent bioinformatics analysis to identify new miRNAs. In total, 126 conserved miRNA belonging to 27 miRNA families were identified, and 34 known but non-conserved miRNAs were also found. Significantly, 72 new potential Amur grapevine-specific miRNAs were discovered. The sequences of these new potential va-miRNAs were further validated through miR-RACE, accumulation of 18 new va-miRNAs in seven tissues of grapevines were also confirmed by real time RT-PCR (qRT-PCR) analysis, and expression levels of va-miRNAs in flowers and berries were basically consistent in identity to those from deep sequenced sRNAs libraries of independent corresponding tissues. We also describe the conservation and variation of va-miRNAs using miR-SNPs and miR-LDs during plant evolution based on comparison of orthologous sequences, and revealed the number and sites of miR-SNP of diverse miRNA families exhibited distinct divergence. Finally, 346 target genes for the new miRNAs were predicted and they include a number of Amur grapevine stress tolerance genes and many genes regulating anthocyanin systhesis and sugar metabolism. Deep sequencing of short RNAs from Amur grapes flowers and fruits identified 72 new potential miRNAs and 34 known but non-conserved miRNAs, indicating that specific miRNAs exist in Amur grapes. These results show that a number of regulatory miRNAs exist in Amur grapes and play an important role in Amur grape growth, development, and response to abiotic or biotic stress. High throughput sequencing was employed to identify miRNAs in Amur grapevine and try to describe their functions in Amur grapevine growth and development.
Project description:we compared the differentially expressed proteins of excretory-secretory (ES) products in three isolates of T. pseudospiralis ML [muscle larvae from Russia (RUS), United States of America (USA) and Australia (AUS)] using isobaric tags for relative and absolute quantification (iTRAQ)-based technology.A total of 2,591 nonredundant proteins were identified, of which 65 (146), 72 (98) and 43 (103) significantly upregulated (downregulated) differentially expressed proteins were detected among pairwise comparisons (T4RUS vs T4USA, T4AUS vs T4USA and T4RUS vs T4AUS). In addition, GO annotation, KEGG and STRING analyses were carried out on the screened differentially altered proteins.
Project description:MicroRNAs (miRNAs) are a class of non-coding RNA molecules which have significant gene regulation roles in organisms. The advent of new high throughput sequencing technologies has enabled the revelation of novel miRNAs. Although there are two recent reports on high throughput sequencing analysis of small RNA libraries from different organs of two grapevine wine varieties, there were significant divergence in the number and kinds of miRNAs sequenced in these studies. More sequencing of small RNA libraries is still important for the discovery of novel miRNAs in grapevine. Here, we initially constructed a small RNA library of flower and fruit tissues of a table grapevine cultivar ‘Summer Black’ and performed sequencing and analysis of sRNAs using the Illumina Solexa platform, expecting to discover more miRNAs related to the development of grapevine flowers and berries and the formation of dessert quality in grapevine berries. Totally, 130 conserved grapevine miRNA (Vv-miRNA) belonging to 28 Vv-miRNA families were validated, and 92 novel potential grapevine-specific ones representing 80 unique ones were first discovered. Forty-two (48.84%) of the novel miRNAs possessed differential semi-quantitative PCR expression profiles in various grapevine tissues that could further confirm their existence in the grapevine, among which twenty were expressed only in grapevine berries, indicating some fruit-specificity. 130 target genes for 46 novel miRNAs could be predicted. The locations of these potential target genes on grapevine chromosomes and their complementary levels with the corresponding miRNAs were also analyzed. Size fractionated small RNAs (16-30 bp) from total RNA extracts was ligated to 5' and 3' adapters, and reverse transcribed. After PCR amplification the sample was subjected to Solexa sequencing. The resultant 35nt sequence data were filtered according to base quality value. The remained sequences were used to trim 5' and 3' adaptors. The clean tags were used for further analysis.
Project description:MicroRNAs (miRNAs) are a class of non-coding RNA molecules which have significant gene regulation roles in organisms. The advent of new high throughput sequencing technologies has enabled the revelation of novel miRNAs. Although there are two recent reports on high throughput sequencing analysis of small RNA libraries from different organs of two grapevine wine varieties, there were significant divergence in the number and kinds of miRNAs sequenced in these studies. More sequencing of small RNA libraries is still important for the discovery of novel miRNAs in grapevine. Here, we initially constructed a small RNA library of flower and fruit tissues of a table grapevine cultivar ‘Summer Black’ and performed sequencing and analysis of sRNAs using the Illumina Solexa platform, expecting to discover more miRNAs related to the development of grapevine flowers and berries and the formation of dessert quality in grapevine berries. Totally, 130 conserved grapevine miRNA (Vv-miRNA) belonging to 28 Vv-miRNA families were validated, and 92 novel potential grapevine-specific ones representing 80 unique ones were first discovered. Forty-two (48.84%) of the novel miRNAs possessed differential semi-quantitative PCR expression profiles in various grapevine tissues that could further confirm their existence in the grapevine, among which twenty were expressed only in grapevine berries, indicating some fruit-specificity. 130 target genes for 46 novel miRNAs could be predicted. The locations of these potential target genes on grapevine chromosomes and their complementary levels with the corresponding miRNAs were also analyzed. Overall design: Size fractionated small RNAs (16-30 bp) from total RNA extracts was ligated to 5' and 3' adapters, and reverse transcribed. After PCR amplification the sample was subjected to Solexa sequencing. The resultant 35nt sequence data were filtered according to base quality value. The remained sequences were used to trim 5' and 3' adaptors. The clean tags were used for further analysis.
Project description:Glycolysis can improve the tolerance of tissue cells to hypoxia, and its intermediates provide raw materials for the synthesis and metabolism of the tumor cells. If it can inhibit the activity of glycolysis-related enzymes and control the energy metabolism of tumor, it can be targeted for the treatment of malignant tumor. The target proteins phosphoglycerate kinase 2 (PGK2), glycerol-3-phosphate dehydrogenase (GPD2) and glucose-6-phosphate isomerase (GPI) were screened by combining transcriptome, proteomics and reverse docking. We detected the binding constant of the active compound using microscale thermophoresis (MST). It was found that esculetin bound well with three potential target proteins. Esculetin significantly inhibited the rate of glycolysis, manifested by differences of cellular lactate production and glucose consumption in HepG2 cells with or without esculetin. It was found that GPD2 bound strongly to GPI, revealing the direct interaction between the two glycolysis-related proteins. Animal tests have further demonstrated that esculetin may have anticancer effects by affecting the activity of PGK2, GPD2 and GPI. The results of this study demonstrated that esculetin can affect the glucose metabolism by binding to glycolytic proteins, thus playing an anti-tumor role, and these proteins which have direct interactions are potential novel targets for tumor treatment by esculetin.
Project description:microRNAs(miRNAs) play critical regulatory roles mainly through cleaving targeted mRNAs or repressing gene translation during plant developments. Grapevine is amongst the most economically important fruit crops with whole genome available, and the study on grapevine miRNAs (Vv-miRNAs) have also been emphasized. However, the regulation mode of Vv-miRNAs on their target mRNAs during grapevine development has not been studied well, especially on a transcriptome-wide level. Here, six small RNA (sRNA) and mRNA libraries from various grapevine tissues were constructed for Illumina and Degradome sequencing. Subsequently, the spatiotemporal variation in the Vv-miRNAs’ regulation on their target genes was systematically analyzed. Totally, 242 known and 132 novel Vv-miRNAs were identified, and 193 target mRNAs including 103 for known and 90 for novel miRNAs were validated in one or more of tissues examined. The interesting finding was that over 50% of novel miRNAs were expressed exclusively in flowers or berries where they had tissue-specific cleavage roles on their target genes, especially, the breadth of their cleavage sites in flower tissues. Moreover, six novel miRNAs in berries were found to response to exogenous gibberellin (GA) and/or ethylene by real time RT-PCR (qRT-PCR) analysis, confirming their regulatory functions during berry development. Other finding was that about 93.6% of the known miRNAs possessed the high conservation in various tissues where their expression levels exhibited some dynamic variations during grapevine development. Significantly, it was found the phenomena that some Vv-miRNA families exist one key member that act as the main regulator of their target genes during grapevine development. Overall design: Six tissues (young leaf, mature leaf, inflorescence, flower, young berry and mature berry) of grapevine were selected, and these samples can be extracted total RNA, and then were separated to small RNAs used for Solexa sequencing, respectively. Subsequenctly, we carried out the analysis of Solexa sequencing data, and performed the research on the dynamic variations of regulatory modes of grapevine miRNAs on their target genes during grapevine development process at the whole transcriptome level.
Project description:Transcriptomic sequencing was performed to obtain the key functional genes involved in the adaptation of oxidative stress induced by hydrogen peroxide (H2O2) in the Arctic bacterium Pseudoalteromonas sp. A2. Exposure to 1 mmol/L H2O2 resulted in large alterations of the transcriptome profile, including significant upregulation of 109 genes and significant downregulation of 174 genes. Functional classification of differentially expressed genes revealed that most of genes affiliated with biological adhesion, negative regulation of biological process, enzyme regulator activity, protein binding transcription factor activity and structural molecular activity were upregulated, and most of genes affiliated with multicellular organismal process and extracellular region were downregulated. It was notably that fifteen genes affiliated with flagella and four genes affiliated with heat shock proteins were significantly upregulated. Meanwhile, nine genes affiliated with cytochrome and cytochrome oxidase, and five genes affiliated with TonB-dependent receptor, were significantly downregulated. However, eighteen genes with antioxidant activity categorized by GO analysis showed differential expressions. This overall survey of transcriptome and oxidative stress-relevant genes can contribute to understand the adaptive mechanism of Arctic bacteria. five significant upregulated genes and five significant downregulated genes were selected using qRT-PCR to cinduct the oxidative stress. overall survey of transcriptomic sequencing by RNA-Seq of the Pseudoalteromonas sp. A2, an isolate from seawater with high activity against H2O2