Altered gene expression associated with thermo-sensitive male sterility in rapeseed (Brassica napus) mutant SP2S. Brassica napus

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.

为揭示温敏雄性不育（temperature sensitive male sterility）潜在的分子机制，本研究对SP2S株系及其近等基因系（Near-Isogenic Line, NIL）SP2F开展了比较转录组分析。本研究通过数字基因表达谱（Digital Gene Expression, DGE）标签测序技术分析了可育与不育植株的转录组差异，鉴定得到大量差异表达与特异性表达的转录本。通过定量RT-PCR，随机选取的10个基因的表达水平与DGE测序数据呈现一致的表达模式。本研究共鉴定得到SP2S株系中827个上调表达转录本与757个下调表达转录本，表明SP2S株系对温度存在复杂的应答反应。SP2S株系中，泛素介导的蛋白水解、苯丙烷生物合成、内吞作用、细胞凋亡、氧化磷酸化、嘌呤代谢、丝裂原活化蛋白激酶（MAPK）信号通路、剪接体及核糖体等重要通路中的部分基因呈现上调表达；而氨酰-tRNA生物合成、基础转录因子、核苷酸切除修复、错配修复与二氧化碳固定等通路中的基因则呈下调表达。此外，参与光合作用、三羧酸循环（citrate cycle, TCA cycle）、磷酸戊糖途径及胁迫应答的部分基因同样存在差异表达。

实验设计：本研究使用两个株系：温敏核不育（Temperature-Sensitive Genic Male Sterile, TGMS）SP2S及其野生型SP2F。SP2S株系源自2007年在自交系SP2中发现的部分雄性不育植株的自发突变体，经多代自交选育获得。可育近等基因系SP2F与SP2S具有完全一致的遗传背景。SP2S与SP2F的种子于9月田间播种，经田间条件下的春化处理后，于12月移栽至温室。温室环境维持温度22℃，光周期长于14小时。采集SP2S与SP2F的幼小花蕾，置于RNAlater保存液中，于-80℃冰箱储存以备后续实验。总RNA采用Trizol®试剂（Invitrogen公司）提取，并通过TRK1001试剂盒（LC Science，美国德克萨斯州休斯顿市）进行纯化。采用Agilent 2100生物分析仪及RNA 6000 Nano LabChip试剂盒（安捷伦，美国）对总RNA的浓度与纯度进行检测。按照Illumina RNA测序（RNA-Seq）实验流程合成并扩增cDNA，随后在Illumina HiSeq2500平台上完成测序。采用Illumina数字基因表达谱标签测序试剂盒，依照厂商说明书，分别以22℃条件下培养的SP2S与SP2F的幼小花蕾总RNA构建DGE文库，每组设置3次生物学重复。按照厂商推荐的实验流程，在Illumina HiSeq2500平台上进行单端测序。对原始测序数据进行过滤，去除包含接头序列、低质量标签及未知碱基N的序列，得到长度为36 nt的干净读段（clean reads），随后对干净读段开展质量评估。采用Bowtie 2软件将所有干净标签比对至本研究组装得到的转录本数据集的单基因（unigene）以及JGI发布的白菜（Brassica rapa）基因组，仅允许1个碱基的错配。为监测双链上的比对事件，本研究的数据收集同时包含正义链与互补反义链序列。计算每个基因对应的完美匹配干净读段的数量，并将其标准化为每百万比对读段中每千碱基外显子模型的读段数（Reads Per Kilobase of exon model per Million mapped reads, RPKM）。基于基因表达水平，以所有样本计数的第三四分位数≥10、p值≤0.05且log2倍变化≥1作为筛选标准，鉴定得到不同样本间的显著差异表达转录本（Differentially Expressed Transcripts, DETs）。采用基因本体（Gene Ontology, GO）分析对DGTs进行功能分类，并通过京都基因与基因组百科全书（KEGG）完成通路富集分析。