Computational protocol: Acetolactate Synthase-Inhibiting Gametocide Amidosulfuron Causes Chloroplast Destruction, Tissue Autophagy, and Elevation of Ethylene Release in Rapeseed

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Protocol publication

[…] Digital gene-expression tag profiling (DGE) is a simple and cost-efficient method to analyse transcriptome using Illumina Solexa technology. Two groups of DGE libraries, each in three biological replicates (DAT1, DAT2, DAT3 and F1, F2, F3), were constructed using the above-mentioned cDNAs of young flower buds with Illumina's Digital Gene Expression Tag Profiling Kit according to the manufacturer's protocol. We performed the single end sequencing on an Illumina GAIIX platform following the vendor's protocol. The adaptor sequences, tags with low quality sequences, and unknown nucleotides N were filtered from the raw data to obtain clean reads with 36 nt in length. The clean reads were deposited in NCBI under Gene Expression Omnibus accession GSE69679. All the reads were aligned with Brassica genome in both JGI database (http://genome.jgi.doe.gov/) and NCBI database (http://www.ncbi.nlm.nih.gov/) by using Bowtie2 v2.1.0 (http://sourceforge.net/projects/bowtie-bio/files/bowtie2/2.1.0/), and only 1 bp mismatch was allowed. We also aligned the clean tags with a previous transcriptome assembly of rapeseed flower buds. The assembly contained 135,702 unigenes (file Trinity_rapeseed.fsa under TSA accession GDFQ00000000), most of which had been successfully annotated in various databases including NCBI non-redundant, JGI, Pfam, SwissProt, and Clusters of Orthologous Groups of proteins (unpublished). The number of perfect reads matching to each unigene was normalized to the number of Reads Per Kilobase of exon model per Million mapped reads (RPKM). The low-frequency transcripts with the 3rd quartile of counts in all samples < 10 were filtered. Based on expression levels, the significant DETs among the two groups of samples were identified with criteria of false discovery rate (FDR) ≤0.05 and fold-change ≥2. The sequence of each DET was searched in NCBI nucleotide collection database by BLAST and the function was annotated by the references in Uniprot (http://www.uniprot.org/) and gene ontology (GO) database (http://www.geneontology.org/). The hierarchical cluster of the DETs was performed by using Cluster 3.0 (http://bonsai.hgc.jp/~mdehoon/software/cluster/software.htm). The GO networks for down-regulated and up-regulated genes were drew by the BiNGO plugin of Cytoscape software (http://www.cytoscape.org/). All possible interactions among the DETs were revealed based on the known and predicted information of Arabidopsis in STRING (https://string-db.org/). [...] Some tissues including upper leaf, small buds of ca. 1 mm length, medium buds of ca. 3 mm, large buds of ca. 5 mm, and anthers of the medium buds were collected from at least 5 plants 3 DAT, with three independent biological replicates for each sample. The RNA was extracted and cDNA was synthesized with 250 ng of total RNA using PrimeScript RT reagent Kit (Takara, Otsu, Japan). Gene-specific primers (Table ) were designed for two function-important ALS loci (ALS1 and ALS3; Zhao et al., ) and 13 selected DETs according to the unigene sequences using the Beacon Designer 7.0 (Bio-Rad, CA, USA). Real-time PCR assays in triplicate were performed using SYBR Green PCR Master Mix (Applied Biosystems, CA, USA) on a QuantStudio 3 thermal cycler (Thermo Fisher Scientific, CA, USA). Tissues from water-treated plants were employed as negative control and B. napus beta-actin7 gene was used as the internal control for data normalization. The relative fold change of each gene in the different samples was calculated by using threshold cycle relative quantification parameter 2−ΔCT (Schmittgen and Livak, ), where ΔCT = (Ctgene - CTactin). Student's t-test was used for pairwise comparisons between the same type of tissues. […]

Pipeline specifications

Software tools Bowtie2, Trinity, BiNGO, Beacon Designer
Databases GEO
Applications RNA-seq analysis, qPCR
Diseases Anemia, Hypochromic, Infertility, Male, Citrullinemia
Chemicals Amino Acids, Chlorophyll, Sulfonylurea Compounds, Pyruvic Acid