ABSTRACT: This study was conducted to determine the magnitude transcript amplification using dTALE/STAP synthetic transcription factor system. It was also conducted to determine whether there was any off-target amplification of endogenous rice genes.
Project description:To gain novel molecular insights into quantitative late blight resistance, we performed a high-resolution quantitative analysis of gene expression using potato cultivars with contrasting SNP alleles at the StAOS2 locus associated with maturity corrected resistance (MCR). SuperSAGE samples were generated from uninfected and infected plants of the selected genotypes under controlled environmental conditions. Genotypes were pooled to reduce the influence of the genetic background on the transcriptome. Nine SuperSAGE samples were prepared from artificially inoculated plants in a growth chamber using total RNA of the pooled 14, 6 and 9 genotypes in groups A1, A2 and B2, respectively, at three infection time points T0, T1 and T2. Combining the tag counts of both NlaIII and DpnII libraries resulted in 1.1 to 6.2 million tags per sample. Of total 266361 unique tags (unitags), 52.6% matched to the potato genome sequence when up to three mismatches per 26 base pairs were allowed, and 23.3% matched without mismatch. Fifteen pair wise comparisons were performed between the nine SuperSAGE samples to identify transcripts that were differentially expressed in response to infection (six comparisons) or between three genotype pools at the infection time points T0, T1 and T2 (nine comparisons). The number of unitags per comparison ranged from 127 000 to 182 000 (average 158 000). Between 2100 and 11800 tags were differentially expressed in pair wise comparisons, depending on arbitrary cut-off p-values for a significant difference. The highest number of differences was observed for the comparison between genotype pools A1 and A2 one day after infection (A1-T1 vs A2-T1), and the lowest between genotype pools A2 and B2 two days after infection (B2-T2 vs A2-T2). The number of differences in response to infection and between genotype pools even before infection (T0) was in the same order of magnitude. Based on the annotations in the DFCI potato gene index, in the potato genome and in few cases by BLASTX searches against the protein database at NCBI, transcripts that showed reproducible differential expression over the infection time course or between genotype pools A1, A2 and B2 were grouped in 16 functional categories, with overlaps between categories. Genes with genotype dependent, constitutive differential expression provide excellent targets for developing novel diagnostic markers for breeding cultivars with improved quantitative resistance to late blight and possibly other biotic and abiotic stresses. Relevant in this respect appear, besides numerous genes of unknown or ill-defined function, genes with known function involved in stress responses, photosynthesis, protein biosynthesis, protein degradation via the 26S proteasome, transport of proteins, lipids, ions and other small molecules, cell wall structure and many others. Eighteen SuperSAGE libraries were constructed based on nine leaf samples from one infection experiment. For each time point (T0, T1 and T2) one leaflet each of 14, 6 and 9 SL genotypes in genotypic groups A1, A2 and B2, respectively, were pooled. Frozen pooled leaf tissue was powdered in a CryoMill. SuperSAGE libraries were generated at GenXPro GmbH (Frankfurt, Germany) essentially as described (Matsumura et al. 2010). To prevent amplification biases the TrueQuant technology was applied as described by B. Rotter (Patent application Nr. WO2009152928). Besides NlaIII (recognition site: 5M-bM-^@M-^Y-CATG-3M-bM-^@M-^Y), DpnII (recognition site: 5M-bM-^@M-^Y-GATC-3M-bM-^@M-^Y) was used as second anchoring enzyme, to capture transcripts without a NlaIII site. Therefore, the 26 bp tags carry either CATG or GATC at their 5M-bM-^@M-^Y end. The libraries were pooled and sequenced by Solexa/Illumina technology (Illumina, Inc., USA).
Project description:Provided data came from a detailed study on Nicotiana benthamiana 16c plants where we use Tobacco Rattle Virus (TRV) as a molecular switch to change the chromatin state of a reporter gene (P35S::GFP) from an actively transcribed to a transcriptionally silenced state. Our approach enables us to interrogate different chromatin states of the same locus with the same set of CRISPR/Cas9 genome editing reagents and systematically describe the effect of chromatin state on the frequency and type of mutations induced at various Cas9 targets in a huge set of independently edited cells.
Project description:Heterosis is an important biological phenomenon; however, the role of small RNA (sRNA) in heterosis of hybrid rice remains poorly described. Here, we performed sRNA profiling of F1 super-hybrid rice LYP9 and its parents using high-throughput sequencing technology, and identified 355 distinct mature microRNAs and trans-acting small interfering RNAs, 69 of which were differentially expressed sRNAs (DES) between the hybrid and the mid-parental value. Among these, 34 DES were predicted to target 176 transcripts, of which 112 encoded 94 transcription factors. Further analysis showed that 67.6% of DES expression levels were negatively correlated with their target mRNAs either in flag leaves or panicles. The target genes of DES were significantly enriched in some important biological processes, including the auxin signalling pathway, in which existed a regulatory network mediated by DES and their targets, closely associated with plant growth and development. Overall, 20.8% of DES and their target genes were significantly enriched in quantitative trait loci of small intervals related to important rice agronomic traits including growth vigour, grain yield, and plant architecture, suggesting that the interaction between sRNAs and their targets contributes to the heterotic phenotypes of hybrid rice. Our findings revealed that sRNAs might play important roles in hybrid vigour of super-hybrid rice by regulating their target genes, especially in controlling the auxin signalling pathway. The above finding provides a novel insight into the molecular mechanism of heterosis. We constructed six sRNA sequencing libraries and six mRNA sequencing libraries of flag leaves and panicles of the super-hybrid rice Liangyou-pei9 (LYP9) combination at the grain-filling stage. The above hybrid rice combination includes F1 hybrid LYP9 and its parental lines including the male-sterile line Peiai64s (PA64s) and the restorer line 93-11.
Project description:The contained data consist of Illumina HiSeq 2500 reads generated from restriction endonuclease digested genomic DNA of Oryza sativa ssp. indica used as proof of concept for plant-RRBS methylome profiling by bioinformatics analyses. Five biological repeats for an inbred control line and a derived epiline of the 4th generation where analysed using two restriction endonuclease combination (MspI-DpnII or MspI-ApekI).
Project description:For most chemicals, environmental toxicity is only investigated for a single generation. In this study we investigate the effect of the fungicide prochloraz across generations in Daphnia magna. The study design included two exposure scenarios; one where all three generations were continuously exposed to prochloraz (100 ug/L) and one where only the first generation (F0) was exposed. We studied effects at different levels of biological organization combining key phenotypic effects, such as growth and reproduction, CYP enzyme activity, metabolomics and for generation F2 also transcriptomics
Project description:The global transcriptional response of Saccharomyces cerevisiae was investigated in low temperature chemostat cultures grown in carbon or nitrogen limitation. During steady state chemostats, the growth rates and in vivo fluxes were kept constant however the growth-limiting nutrient was significantly higher at 12oC than at 30oC and had significant effects on transcriptional responses. Growth at 12oC resulted in a rearrangement of transporters for the limiting nutrient, where hexose transporters (HXTs) and ammonium permeases (MEPs) were differentially expressed in cultures grown at 30oC in carbon and nitrogen limitations, respectively. In addition, we found repression of genes encoding proteins in reserve carbohydrates metabolism and metabolism of alternative carbon or nitrogen sources other than glucose or ammonia. However, there were also similar responses when the transcriptional response was evaluated regardless of the growth-limiting nutrient. In particular, induction of ribosome biogenesis genes emphasizes the significance of transcription and translational adaptation at low temperature. In contrast, genes encoding proteins during stress response were downregulated. This down-regulation of stress elements better known as environmental stress response (ESR) is in contradiction with previous low temperature transcriptome analyses. During continuous steady state low temperature cultivation, ESR no longer plays an integral role in S. cerevisiaeM-bM-^@M-^Ys response to temperature change. Similarly, trehalose accumulation, consistent with its gene expression, was not indispensable for growth at 12oC. This response, however, does not exclude that ESR may be required for transition phase in low temperature growth when cells are transferred from one temperature to another. Keywords: chemostat temperature 12 degree celsuis 30 degree celsius The global transcriptional response of Saccharomyces cerevisiae was investigated in low temperature chemostat cultures grown in carbon or nitrogen limitation at a dilution rate of 0.03h-1
Project description:A NimbleGen array containing both gene-based and RJM repeat junction probe sequences derived from Ae. Tauschii was developed and used to map the Chinese Spring nullisomic-tetrasomic lines and deletion bin lines of the D genome chromosomes. The NimbleGen array was hybridized in duplicate with Cy3 labeled seven nullisomic-tetrasomic lines, deletion bin lines for D genome chromosomes, and control reference Chinese Spring; as well as Cy5 labeled reference line Chinese Spring.
Project description:The experiment looks for diurnal-regulated genes in Medicago truncatula plants. Medicago truncatula Jester accession plants were entrained for18 days in long days (16h light:8h dark) at 24°C constant temperature. Samples for two biological replicates per time point were collected every 4h for 24h beginning at ZT0 (subjective dawn) until ZT20. Total RNA was extracted from ground tissue using an RNeasy RNA extraction kit (Qiagen). Extracted total RNA was DNAse-treated (Invitrogen). Total RNA (2-3ug) was dried down and preserved in Sigma-Aldrich RNAstable before being couriered to BGI Tech Solutions (HONGKONG) Co., Ltd (https://www.bgi.com/global). BGI Tech prepared and processed libraries for 20M PE100 reads using their DNBseq platform - BGISEQ-500.