Unknown,Transcriptomics,Genomics,Proteomics

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Cryptochrome 1 regulates growth and development in Brassica through alteration in the expression of genes involved in light, phytohormone and stress signalling.

ABSTRACT: To cover a comprehensive understanding of the complex regulatory networks controlling cryptochrome 1(CRY1)-mediated photomorphogenic response in Brassica, a genome-wide differential expression analysis was performed for the 5-day-old T3 BnCRY1-OE and wild type seedlings grown under white light (100 M-BM-5mol mM-bM-^@M-^S2sM-bM-^@M-^S1) on 0.8% agar-gelled MS (Murashige and Skoog) medium containing 2% sucrose. Total RNA was extracted and cleaned up with the Qiagen RNeasy Plant Mini Prep Kit as per the manufacturerM-bM-^@M-^Ys instructions (Qiagen). The yield and RNA purity were determined spectrophotometrically. Integrity of the RNA was checked using Agilent Bioanalyzer (Agilent Technologies, USA). 500 ng RNA of each of the sample was processed for microarray analysis according to Agilent protocol. Keywords: Cryptochrome, Brassica, photomorphogenesis, differential expression. Agilent one-color experiment, Organism: B. napus (Brassica) Oligo Microarray, Agilent-022520 B. napus (Brassica) Oligo Microarray 4x44K, Labeling kit: Agilent Quick-Amp labeling Kit (p/n5190-0442)

ORGANISM(S): Brassica juncea

SUBMITTER: Pooja Sharma

PROVIDER: E-GEOD-26797 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Cryptochrome 1 regulates growth and development in Brassica through alteration in the expression of genes involved in light, phytohormone and stress signalling.

Sharma Pooja P Chatterjee Mithu M Burman Naini N Khurana Jitendra P JP

Plant, cell & environment 20131114 4

The blue light photoreceptors cryptochromes are ubiquitous in higher plants and are vital for regulating plant growth and development. In spite of being involved in controlling agronomically important traits like plant height and flowering time, cryptochromes have not been extensively characterized from agriculturally important crops. Here we show that overexpression of CRY1 from Brassica napus (BnCRY1), an oilseed crop, results in short-statured Brassica transgenics, likely to be less prone to ...[more]

PMID: 24117455

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Project description:In this study, time dependent (4 h to 96 h) transcriptome changes in roots and shoots of Brassica juncea were analyzed under arsenate (AsV) stress by using Agilent platform. A total of 1285 genes showed significant change in expression pattern upon arsenate (AsV) exposure. The genes belonged to various signaling pathways including hormones (jasmonate, abscisic acid (ABA), auxin and ethylene) and kinases. Significant effects were also noticed on genes of sulfur, nitrogen, CHO, and lipid metabolisms along with photosynthesis. These studies indicated interconnections among sulfur metabolism, jasmonate and kinase signaling pathways. Transcriptome results and biochemical analyses highlight that signaling and metabolic pathways work in a dynamic coordination to perceive and respond to the stress. A set of 53939 sequences, which include EST and nucleotide sequences of Brassica sp., were downloaded from GenBank. These sequences were subjected to clustering and analyzed using CAP3/tgicl program and other in-house tools. A set of 26881 non-redundant sequences was created, which was used for probe design. This set included 1720 Brassica juncea, 15259 Brassica napus and 5075 Brassica rapa sequences; and 6456 from other species. Probes were designed for the non-redundant sequences using Agilent eArray tool. Best probe composition algorithm with the option to design multiple probes for each sequence was the main criteria used for the probe design. AgilentM-bM-^@M-^Ys 4x44 array was selected to print the oligos. A set of positive and negative controls were also added on to the microarray chip. A set of replicate probes were also added for calculating the intra-array reproducibility. The quantity and purity of the RNA was determined by absorbance at 260 nm and 260/280 absorbance ratio respectively. Each of the total RNA preparations was individually assessed for RNA quality based on the 28S/18S ratio and RIN measured on an Agilent 2100 Bioanalyzer system using the RNA 6000 Nano LabChip Kit .With the use of AgilentM-bM-^@M-^Ys 1-Color Quick Amp Labeling Kit, 500 ng of high quality total RNA was denatured in the presence of a T7 promoter primer and a 1-Color RNA Spike-In Kit. Reverse transcriptase was used to reverse-transcribe the mRNA. cDNA was used as a template for in vitro transcription where a T7 RNA polymerase simultaneously amplified target material and incorporated cyanine 3-labeled CTP. Labeled cRNA was purified using spin columns from the Qiagen RNeasy Mini Kit and the quantity and quality of the cRNA was determined by Nanodrop ND-1000 UV-VIS spectrophotometer. With the use of AgilentM-bM-^@M-^Ys Gene Expression Hybridization Kit, 1.65 M-BM-5g of cyanine 3-labeled linearly amplified cRNA was added to a hybridization cocktail and then fragmented. The hybridization cocktail was then dispensed into the wells of gasket slides on top of which a Brassica 4x44K Gene Expression Microarray was placed. This microarray M-bM-^@M-^\sandwichM-bM-^@M-^] was then sealed in a hybridization chamber. The microarrays were hybridized at 65M-BM-0C rotating at 10 RPMs for 17 hours. Slides were washed in AgilentM-bM-^@M-^Ys Gene Expression Wash Buffers according to manufacturer specifications. The microarrays were scanned on the Agilent Microarray Scanner. Scanner data extraction and analysis was performed using Agilent Technologies Feature Extraction software version 10.7.3.1.Microarray data quality was evaluated by reviewing ten standard QC metrics generated by the Feature Extraction Software [Consult the Agilent Technologies Feature Extraction Software version 10.7.3.1 reference guide for a full explanation of the QC metrics]. To determine if there was a large hybridization, staining, or wash artifacts, a visual examination of each array was performed by loading the array image into Feature Extraction Software. Microarrays were determined to be free of any large artifacts (M-bM-^IM-% 10% of total surface area) that could have affected the quality of the data.

Dataset Information

Cryptochrome 1 regulates growth and development in Brassica through alteration in the expression of genes involved in light, phytohormone and stress signalling.

Publications

Cryptochrome 1 regulates growth and development in Brassica through alteration in the expression of genes involved in light, phytohormone and stress signalling.

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