National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Technology, Beijing Forestry University, Tsinghua East Road 35, Haidian District, Beijing 100083, PR China.
Key Laboratory of Tropical Forest Ecology, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Germplasm Bank, Mengla, 666303 Yunnan, PR China.
Plant Physiol Biochem. 2014 Oct;83:316-26. doi: 10.1016/j.plaphy.2014.08.001. Epub 2014 Aug 12.
To investigate regulatory processes and protective mechanisms leading to desiccation tolerance (DT) in seeds, cDNA amplified fragment length polymorphism (cDNA-AFLP) in conjunction with 128 primer combinations was used to detect differential gene expression in rape seeds in response to DT during seed development and germination. We obtained approximately 8000 transcript-derived fragments (TDFs), of which 394 TDFs with differential expression patterns ("sustained expression", "up-regulated", "couple with seed DT", and "down-regulated") were excised from gels and re-amplified by polymerase chain reaction (PCR). After sequencing and comparison with the National Center for Biotechnology Information database, 176 TDFs presented significant similarity with known genes that could be classified into the following categories: metabolism and energy, stress resistance and defense, storage, signal transduction, and other functional categories. Using semiquantitative reverse-transcription PCR and real-time PCR approaches, the significance of the differences was further confirmed in fresh seeds and dehydrated seeds. The genes that encode superoxide dismutase, peroxiredoxin, caleosin, oleosin S3, steroleosin, late embryogenesis abundant protein, glutathione reductase, β-glucosidase, S23 transcriptional repressor, and some heat-shock proteins could be associated with DT. The results of this study will aid in the identification of candidate genes for future experiments that seek to understand seed DT.
为了研究导致种子耐旱性（DT）的调控过程和保护机制，我们使用 cDNA 扩增片段长度多态性（cDNA-AFLP）结合 128 对引物组合，检测了油菜种子在种子发育和萌发过程中对 DT 的响应中的差异基因表达。我们获得了大约 8000 个转录衍生片段（TDFs），其中 394 个 TDFs 的表达模式存在差异（“持续表达”、“上调”、“与种子 DT 偶联”和“下调”），从凝胶中切下并通过聚合酶链反应（PCR）重新扩增。测序并与国家生物技术信息中心数据库进行比较后，176 个 TDFs 与已知基因具有显著相似性，可分为以下几类：代谢和能量、应激抗性和防御、储存、信号转导和其他功能类别。使用半定量逆转录 PCR 和实时 PCR 方法，在新鲜种子和脱水种子中进一步证实了差异的显著性。编码超氧化物歧化酶、过氧化物酶、钙调素、油质体 S3、甾醇结合蛋白、晚期胚胎丰富蛋白、谷胱甘肽还原酶、β-葡萄糖苷酶、S23 转录阻遏物和一些热休克蛋白的基因可能与 DT 有关。本研究的结果将有助于鉴定候选基因，为未来理解种子 DT 的实验提供帮助。
