Zhou Xue, Liu Zhiyong, Ji Ruiqin, Feng Hui
Department of Horticulture, Shenyang Agricultural University, Shenyang, 110866, People's Republic of China.
Mol Genet Genomics. 2017 Oct;292(5):967-990. doi: 10.1007/s00438-017-1324-2. Epub 2017 May 10.
We studied the underlying causes of multiple-allele-inherited male sterility in Chinese cabbage (Brassica campestris L. ssp. pekinensis) by identifying differentially expressed genes (DEGs) related to pollen sterility between fertile and sterile flower buds. In this work, we verified the stages of sterility microscopically and then performed transcriptome analysis of mRNA isolated from fertile and sterile buds using Illumina HiSeq 2000 platform sequencing. Approximately 80% of ~229 million high-quality paired-end reads were uniquely mapped to the reference genome. In sterile buds, 699 genes were significantly up-regulated and 4096 genes were down-regulated. Among the DEGs, 28 pollen cell wall-related genes, 54 transcription factor genes, 45 phytohormone-related genes, 20 anther and pollen-related genes, 212 specifically expressed transcripts, and 417 DEGs located on linkage group A07 were identified. Six transcription factor genes BrAMS, BrMS1, BrbHLH089, BrbHLH091, BrAtMYB103, and BrANAC025 were identified as putative sterility-related genes. The weak auxin signal that is regulated by BrABP1 may be one of the key factors causing pollen sterility observed here. Moreover, several significantly enriched GO terms such as "cell wall organization or biogenesis" (GO:0071554), "intrinsic to membrane" (GO:0031224), "integral to membrane" (GO:0016021), "hydrolase activity, acting on ester bonds" (GO:0016788), and one significantly enriched pathway "starch and sucrose metabolism" (ath00500) were identified in this work. qRT-PCR, PCR, and in situ hybridization experiments validated our RNA-seq transcriptome analysis as accurate and reliable. This study will lay the foundation for elucidating the molecular mechanism(s) that underly sterility and provide valuable information for studying multiple-allele-inherited male sterility in the Chinese cabbage line 'AB01'.
我们通过鉴定可育和不育花芽之间与花粉不育相关的差异表达基因（DEG），研究了大白菜（Brassica campestris L. ssp. pekinensis）多等位基因遗传雄性不育的潜在原因。在这项工作中，我们通过显微镜观察验证了不育阶段，然后使用Illumina HiSeq 2000平台测序对从可育和不育芽中分离的mRNA进行转录组分析。约2.29亿条高质量双端 reads 中约80% 被唯一映射到参考基因组。在不育芽中，699个基因显著上调，4096个基因下调。在差异表达基因中，鉴定出28个花粉细胞壁相关基因、54个转录因子基因、45个植物激素相关基因、20个花药和花粉相关基因、212个特异性表达转录本以及417个位于连锁群A07上的差异表达基因。六个转录因子基因BrAMS、BrMS1、BrbHLH089、BrbHLH091、BrAtMYB103和BrANAC025被鉴定为假定的不育相关基因。由BrABP1调控的弱生长素信号可能是导致此处观察到的花粉不育的关键因素之一。此外，本研究还鉴定出了几个显著富集的GO术语，如“细胞壁组织或生物合成”（GO:0071554）、“膜内在成分”（GO:0031224）、“膜整合成分”（GO:0016021）、“作用于酯键 的水解酶活性”（GO:0016788），以及一条显著富集的途径“淀粉和蔗糖代谢”（ath00500）。qRT-PCR、PCR和原位杂交实验验证了我们的RNA-seq转录组分析准确可靠。本研究将为阐明不育的分子机制奠定基础，并为研究大白菜品系‘AB01’中的多等位基因遗传雄性不育提供有价值的信息。
