Project description:MicroRNAs (miRNA) are ~21 nucleotide long, small endogenous non-coding RNAs that functioning in regulation of gene expression found in many eukaryotes. In this study, small RNA libraries of opium poppy from four different tissues (leaf, root, capsule, stem) were sequenced using high-throughput next generation Illumina sequencing (Solexa) technology to investigate potential mode of actions of miRNAs in alkaloid biosynthesis. A total of 27 opium poppy miRNAs which have roles in regulation of alkaloid biosynthesis were identified in this study. A six chip study using miRNA isolated from four separate tissues (capsule, leaf, stem, root). small RNA libraries of opium poppy tissues were sequenced using high-throughput next generation Illumina sequencing (Solexa) technology to investigate potential mode of actions of miRNAs in alkaloid biosynthesis. Furthermore, the novel opium poppy miRNAs were also confirmed by a direct small RNA cloning strategy. The microarray platform were performed to measure and analyze the mirnome of the different opium poppy tissues.
Project description:MicroRNAs (miRNA) are ~21 nucleotide long, small endogenous non-coding RNAs that functioning in regulation of gene expression found in many eukaryotes. In this study, small RNA libraries of opium poppy from four different tissues (leaf, root, capsule, stem) were sequenced using high-throughput next generation Illumina sequencing (Solexa) technology to investigate potential mode of actions of miRNAs in alkaloid biosynthesis. A total of 27 opium poppy miRNAs which have roles in regulation of alkaloid biosynthesis were identified in this study.
Project description:Tobacco, as an important cash crop and model plant, has been studied and explored in various aspects. In China, Yunyan 87 was recognized as a flue-cured tobacco variety and had been widely concerned due to its excellent product quality characteristics. The quality of tobacco products depends on the compound collection of tobacco leaves, including pigments, carbohydrates, amino acids, polyphenols and alkaloids. Present study investigated tobacco seedlings, with the assistant of the untargeted metabonomic technology and the label-free proteomic technology to analyze metabolites and proteins differences in leaf, stem, and root groups respectively. From 298 metabolites and 4993 proteins obtained, there were significant differences in both primary and secondary metabolism involved aroma precursors biosynthesis in seedling tobacco leaves, stems, and roots, such as carbohydrate metabolism, energy metabolism, and amino acid biosynthesis, and secondary metabolism phenylpropanoids, flavonoids and alkaloid biosynthesis in this study. Especially alkaloids metabolites identification results showed nornicotine, anatabine, anatalline, and myosmine, were significantly higher in tobacco roots than in leaves, and stems at seedling stage.
Project description:Identifying the intracellular and cell wall-ionically bound glycoside hydrolases (GHs) carbohydrate esterases (CEs) at the late growth stage of Arabidopsis stems is important for understanding the mechanisms regulating the cell wall integrity. The fact that whole plant stems are used as biomass feedstocks and the mixing of intracellular proteins with the cell wall proteome caused by an increasing proportion of broken cells of stems at the late growth stage pose a challenge in identifying these GHs and CEs. Here, we used a CaCl2-extraction procedure to isolate non-structural proteins from Arabidopsis whole stems, followed by protein solution method for direct identification of stem proteins, and by SDS-PAGE separation and protein gel band method for improving the identification of stem proteins, using Nano-LC-MS/MS analysis. Totally, 75 and 236 stem proteins were identified by using these two methods, respectively, confirming the later method (i.e. protein gel method) identified three time more stem proteins. Among these proteins, based on cell wall protein databases and data mining analyses, 6 and 22 proteins were identified as cell wall proteins from the late growth stage Arabidopsis stems, by using these two methods respectively.
Project description:Identifying the intracellular and cell wall-ionically bound glycoside hydrolases (GHs) carbohydrate esterases (CEs) at the late growth stage of Arabidopsis stems is important for understanding the mechanisms regulating the cell wall integrity. The fact that whole plant stems are used as biomass feedstocks and the mixing of intracellular proteins with the cell wall proteome caused by an increasing proportion of broken cells of stems at the late growth stage pose a challenge in identifying these GHs and CEs. Here, we used a CaCl2-extraction procedure to isolate non-structural proteins from Arabidopsis whole stems, followed by protein solution method for direct identification of stem proteins, and by SDS-PAGE separation and protein gel band method for improving the identification of stem proteins, using Nano-LC-MS/MS analysis. Totally, 75 and 236 stem proteins were identified by using these two methods, respectively, confirming the later method (i.e. protein gel method) identified three time more stem proteins. Among these proteins, based on cell wall protein databases and data mining analyses, 6 and 22 proteins were identified as cell wall proteins from the late growth stage Arabidopsis stems, by using these two methods respectively.
Project description:Tobacco with modified genetics controlling alkaloid accumulation has been of interest because of the possibility of mandated lowering of nicotine levels in combustible cigarettes by regulatory authorities. Transcription factors coded by the Nic1, Nic2, and Myc2a loci act as positive regulators of genes directly involved in alkaloid accumulation. Different combinations of alternative alleles at these loci were assembled in homozygous nearly isogenic lines (NILs). Recessive nic1/nic2 alleles were found to have greater influence on alkaloid reduction as compared to a mutant myc2a allele. When combined into single genotypes, these alleles operated in an additive manner to further reduce alkaloid levels in field and greenhouse experiments. This was at the expense of reduced cured leaf quality, however. An RNA-seq experiment was carried out to investigate global changes in root expression due to different genotypes at these three loci. Up to 681 differentially expressed genes (DEGs) were identified between the studied NILs, with expression of most DEGs being downregulated by recessive alleles. In general, the mutant myc2a allele appeared to suppress a subset of previously characterized alkaloid biosynthetic genes with relatively weaker effect as compared to the nic1/nic2 alleles. There was little evidence that root expression of Nic1 or Nic2 genes was affected by allelic variability at the Myc2a locus, or vice versa. The list of DEGs influenced by genotypes at these three loci may contain candidate genes coding for currently uncharacterized enzymes involved with tobacco alkaloid accumulation.
Project description:This dataset belongs to a set of three RNA-Seq experiments that were carried out to study the regulation of monoterpenoid indole alkaloid production in the medicinal plant Catharanthus roseus. For this dataset, C. roseus stems were dissected to separate the epidermis from the lower tissues. Leaves were dissected to obtain veins and veinless leaves. As controls, undissected leaves and stems were used. Three biological replicates were analyzed per sample.