Computational protocol: Genome-Wide Analysis of the PYL Gene Family and Identification of PYL Genes That Respond to Abiotic Stress in Brassica napus

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[…] To further characterize the different temporal and spatial gene expression patterns of the BnPYL gene family, we analyzed RNA sequencing (RNA-seq) data. Transcriptome sequencing datasets were deposited in the BioProject ID PRJNA358784, which was used to perform RNA-seq of different B. napus cultivar ZS11 tissues. We analyzed the total RNA-seq data of the roots, stems, leaves, flowers, seeds, and siliques at the germination, seedling, bud, initial flowering, and full-bloom stages. We quantified these gene expression levels on the basis of their fragments per kilobase of exon per million reads mapped (FPKM) values using Cufflinks with default parameters [], and represented these results using HemI 1.0 software []. To further understand the functions of these genes, we used BLAST software (https://blast.ncbi.nlm.nih.gov/) to align the BnPYL sequences with entries in the NCBI nonredundant (NR) protein [], Swiss-Prot [], GO [], clusters of orthologous groups (COG) [], eukaryotic orthologous groups (KOG) [], Protein family (Pfam) [], and Kyoto encyclopedia of genes and genomes (KEGG) databases [], and conducted GO enrichment analysis of those BnPYLs that were annotated. GO enrichment was performed using the BinGO program of Cytoscape_3.4.0 software [] with an FDR < 0.01. [...] RNA was extracted from drought-, heat- and salinity-treated samples using an EZ-10 DNAaway RNA Mini-prep Kit (Sangon Biotech, Shanghai, China), according to the manufacturer’s instructions. RNA concentrations were measured by a NanoDrop 2000 (Thermo Fisher Scientific, Worcester, MA, USA), and RNA integrity was evaluated by electrophoresis. One microgram of RNA template from each sample was used to synthesize the first-strand of complementary DNA (cDNA) using an iScriptTM cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA), and the cDNA solution was then diluted 20 times with distilled deionized water. Each reaction had a final volume of 20 µL and contained 2 µL of 20-fold-diluted cDNA solution, 10 µL of SYBR® Green Supermix (Bio-Rad), 0.4 µL of 10 mM forward and reverse primers, and 7.2 µL of distilled deionized water. We performed qRT-PCR on a CFX96 Real-time System (Bio-Rad), according to the manufacturer’s instructions. The qRT-PCR program was as follows: 98 °C for 30 s, then 40 cycles of 98 °C for 15 s, 60 °C for 30 s, and an increase from 65 °C to 95 °C with an increment of 0.5 °C every 0.05 s. Three technical replications were performed per sample. We calculated the relative gene expression levels based on the 2−ΔΔCt method using Actin7 of B. napus as an internal control for normalizing gene expression levels. RT-PCR primers were designed on Primer Premier Software (version 5.0). Given the highly homologous candidates in B. napus, all of the primer sequences avoided false priming and are listed in . All of the qRT-PCR results were displayed using HemI 1.0 software []. […]

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