Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptional analysis of epithelial cells in grapefruit peel

ABSTRACT: Epithelial cells (EC) lining the most inner layers of secretory glands in Citrus peel are hypothesized to be the specialized cells that synthesize citrus essential oil. The major biosynthetic pathway(s) for essential oil are therefore postulated to be specifically and highly active in EC; transcripts that are involved in the pathway(s) are expected to be highly up-regulated in the cells as well. We performed cell-specific transcriptional analysis using GeneChip Citrus Genome Arrays to probe the global gene expression in EC during initial stage of essential oil biosynthesis and to identify groups of highly expressed genes in the EC. Grapefruits of two different sizes (diameters at 28 mm and 41 mm), at which points where biosynthesis of essential oil entering its active phase, were selected for the purpose of this experiment. For comparison, we selected parenchyma cells (PC), the non-oil-biosynthesizing cells, as the control cell type. Grapefruit peels were fixed, embedded in paraffin, sectioned and mounted on slides prior to cells isolation using laser microdissection and pressure catapulting. RNA was extracted from the isolated cells and then used for microarray hybridization. Transcriptional analysis were carried out by comparing transcript profiles between different cell types of the same time points, and same cell type at the different time points.

ORGANISM(S): Citrus x paradisi

SUBMITTER: Siau Sie Voo

PROVIDER: E-GEOD-33964 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Huanglongbing (HLB) is a worldwide devastating disease of citrus. There are no effective control measures for this newly emerging but century-old disease. A powerful oligonucleotide microarray of high-density 16S rRNA genes, the PhyloChip microarray, has been developed and effectively used to study bacterial diversity, especially from environmental samples. In this article, we aim to decipher the bacterial microbiome in HLB-affected citrus versus non-infected citrus as well as in citrus plants treated with ampicillin and gentamicin using PhyloChip-based metagenomics. The antibiotic treatments were conducted on the randomized complete block design with three replicates. For each replicate, 15 scions were treated in each antibiotic treatment (Amp and Gm) and control (CK1 and CK2). HLB-affected budsticks were sampled from severely HLB-affected field rough lemons (cv. Lemon #76) at the USDA-ARS-USHRL farm in Fort Pierce, FL and tested positive for Las by real-time qPCR. They were soaked in the antibiotic treatments; ampicillin sodium at a concentration of 1.0 g/L (Amp, Sigma-Aldrich, St. Louis, MO) or gentamicin sulfate at a concentration of 100 mg/L (Gm, Sigma-Aldrich, St. Louis, MO) and water as the diseased control (CK1), overnight in a fume hood under ventilation and lighting. Las-free budsticks, which tested negative by qPCR from healthy rough lemons, were also soaked in water as the healthy control (CK2). The budsticks were grafted onto two-year-old healthy grapefruit (Citrus paradisi 'Duncan') rootstocks and covered using plastic tape for three weeks. To improve scion growth, new flush from the rootstocks was removed after grafting and then allowed to grow. All experimental plants were grown in an insect-proof greenhouse. The first leaf samples from scions (rough lemon) and rootstocks (grapefruit) for DNA extraction were taken four months after inoculation, and second samplings were taken at six month after inoculation. The leaves were washed in tap water and then rinsed three times with sterile water. The midribs of the leaves were excised, frozen in liquid nitrogen, and stored at -80M-BM-0C. The midribs of five leaves from each sample were pooled, and DNA was isolated for qPCR analysis for Las bacterium. DNA from the leaf midribs of scions for the PhyloChipT G3 analysis, which was extracted from all samples of the same treatment, was pooled in equal amounts and quantified by the PicoGreenM-BM-. method. The PhyloChipTM G3 analysis was conducted by Second Genome Inc. (San Francisco, CA).

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Transcriptional analysis of epithelial cells in grapefruit peel

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