Transcriptome profile of Brassica napus seed development

We profiled the gene regulatory landscape of Brassica napus reproductive development using RNA sequencing. Comparative analysis of this nascent allotetraploid across the plant lifecycle revealed the contribution of each subgenome to plant reproduction. Global mRNA profiling across reproductive development revealed lower accumulation of C subgenome transcripts relative to the A subgenome. Subgenome-specific transcriptional networks identified distinct transcription factor families enriched in each of the A and C subgenome in early seed development. Analysis of a tissue specific transcriptome of early seed development revealed transcription factors predicted to be regulators encoded by the A subgenome are expressed primarily in the seed coat whereas regulators encoded by the C subgenome were expressed primarily in the embryo. Whole genome transcription factor networks identified BZIP11 as an essential regulator of early B. napus seed development. Knockdown of BZIP11 using RNA interference resulted in knockdown of predicted target genes, and a reproductive-lethal phenotype. Our data indicate that subgenome bias are characteristic features of the B. napus seed throughout its development, and that such bias might not be universal across the embryo, endosperm, and seed coat of the developing seed. We also find that examining transcriptional networks spanning both the A and C genomes of the whole B. napus seed can identify valuable targets for seed development research. We suggest that-omics level approaches to studying gene regulation in B. napus can benefit from both broad and high-resolution analyses. RNA were isolated from ovules, and seeds 7 (GLOB), 10 (HEART), 28 (MG), and 35 (DS) days post fertilization. Three replicates were sequenced for each stage except DS, for which only 2 replicates were sequenced. Samples were sequenced 100bp SE reads on the Illumina HiSeq2500. Reads were trimmed with Trimmomatic 0.36 and aligned with HiSat2 to the DH12075 genome. Counts, normalization, and differential expression were called using Cuffquant and Cuffdiff.

本研究利用RNA测序（RNA sequencing）解析了甘蓝型油菜（Brassica napus）生殖发育的基因调控全景。对该新生异源四倍体（allotetraploid）的全生命周期开展比较分析，明确了各亚基因组（subgenome）对植物生殖过程的贡献。生殖发育全过程的全局mRNA谱分析显示，相较于A亚基因组（A subgenome），C亚基因组（C subgenome）的转录本积累水平更低。通过亚基因组特异性转录调控网络分析，鉴定出在种子早期发育阶段，A、C亚基因组分别富集有独特的转录因子（transcription factor）家族。对种子早期发育的组织特异性转录组分析显示，由A亚基因组编码的、被预测为调控因子的转录因子主要在种皮（seed coat）中表达，而由C亚基因组编码的调控因子则主要在胚（embryo）中表达。全基因组转录因子网络分析鉴定出BZIP11是甘蓝型油菜早期种子发育的关键调控因子。利用RNA干扰（RNA interference）敲低BZIP11的表达，可导致其预测靶基因的表达量下调，并引发生殖致死表型（reproductive-lethal phenotype）。本研究数据表明，亚基因组偏好性（subgenome bias）是甘蓝型油菜种子整个发育过程的特征，且该偏好性在发育种子的胚、胚乳（endosperm）和种皮中并非普遍存在。此外，对甘蓝型油菜全种子的A、C基因组转录调控网络进行分析，可为种子发育研究鉴定出有价值的靶标基因。我们认为，针对甘蓝型油菜基因调控的组学（-omics）水平研究方法，可同时结合广谱性与高分辨率分析策略以获得更佳效果。从受精后7天（GLOB）、10天（HEART）、28天（MG）及35天（DS）的胚珠和种子中提取RNA；除DS阶段仅设置2次生物学重复外，其余每个阶段均设置3次生物学重复。采用Illumina HiSeq2500平台对样本进行100bp单端测序（Single-end, SE）。测序reads通过Trimmomatic 0.36进行质量修剪，并利用HiSat2比对至DH12075参考基因组。基因计数、标准化及差异表达分析通过Cuffquant与Cuffdiff完成。