GSE69638
Altered gene expression associated with thermo-sensitive male sterility in rapeseed (Brassica napus) mutant SP2S
To reveal the possible molecular mechanism underpinning the temperature sensitive male sterility, a comparative transcriptome analysis of the SP2S line and its NIL SP2F was conducted. Transcriptome differences between fertile and sterile plants were analyzed by using digital gene expression (DGE) tag profiling, and numerous differentially and specifically expressed transcripts were identified. By quantitative RT-PCR, expression levels of 10 genes randomly selected showed consistent expression patterns with the DGE data. A total of 827 up-regulated and 757 down-regulated expression transcripts in SP2S were identified, indicating  the complex responses to temperature in the SP2S line. Some genes in important pathways including ubiquitin mediated proteolysis, phenylpropanoid biosynthesis, endocytosis, apoptosis, oxidative phosphorylation, purine metabolism, MAPK signaling, spliceosome,  and ribosome were up-regulated in SP2S while other genes in the pathways of aminoacyl-tRNA biosynthesis, basal transcription factors, nucleotide excision repair, mismatch repair, and CO2 fixation were down-regulated. Some genes involved in photosynthesis, citrate cycle (TCA cycle), pentose phosphate, and stress-responsive were also differentially expressed. Overall design: Two lines named TGMS SP2S and the wild-type SP2F were used in this study. SP2S was bred after consecutive generations of selfing from spontaneous mutation of partially male-sterile plants found in an inbred line, SP2, in 2007. The fertile NIL (near-isogenic line) SP2F had the same genetic background with SP2S. The seeds of SP2S and SP2F were sown in field in September and the seedlings were transplanted into a greenhouse in December after vernalization under field condition. The temperature was kept at 22°C and photoperiod was longer than 14h. Young floral buds from SP2S and SP2F were collected, put into RNAlater, and stored at -80°C until further processing. Total RNAs were extracted using the Trizol® Reagent (Invitrogen), and purified using TRK1001 Kit (LC Science, Houston, TX). The total RNA quantity and purity were analyzed by using of Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, USA). The cDNAs were amplified according to the Illumina RNA-Seq protocol and sequenced on Illumina Hiseq2500 platform. Two groups of DGE libraries, each in three biological replicates, were constructed using total RNA of young floral buds of SP2S and SP2F grown at 22 Celsius degree with Illumina's Digital Gene Expression Tag Profiling Kit according to the manufacturer's protocol. We performed the single end sequencing on an Illumina Hiseq2500 following the vendor's recommended protocol. The raw data containing adaptor sequences, tags with low quality sequences and unknown nucleotides N were filtered to obtain clean reads with 36 nt in length. Clean reads were then conducted for quality assessment. All clean tags were mapped to the unigenes of our assembled transcripts dataset and JGI Brassica rapa genome by Bowtie 2 software, only 1 bp mismatch is allowed. For monitoring the mapping events on both strands, both the sense and the complementary antisense sequences were included in the data collection. The number of perfect clean reads corresponding to each gene was calculated and normalized to the number of Reads Per Kilobase of exon model per Million mapped reads (RPKM). Based on the expression levels, the significant DETs (Differentially expressed transcripts) among different samples were identified with the 3rd quartile of counts in all samples = 10, p value =0.05, and log2 fold-change=1. Gene ontology (GO) analysis was conducted for functional classification of DGTs and pathway analysis was carried out by using KEGG.