Ye Rui, Yao Quan-Hong, Xu Zhi-Hong, Xue Hong-Wei
National Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 200032 Shanghai, China.
Plant J. 2004 Apr;38(2):348-57. doi: 10.1111/j.1365-313X.2004.02037.x.
Summary An efficient yeast-based system was developed for the isolation of plant cDNAs encoding transcription factors (TFs) and proteins with transcription activation functions (co-activators). The system consists of two vectors: (i) a reporter vector (pG221) harboring the iso-1-cytochrome c (CYC1) core promoter and the beta-galactosidase (lacZ) gene; and (ii) a cDNA library construction vector (pYF503), which yields a library of plant peptides fused to the GAL4-binding domain (GAL4-BD). Expression of a peptide harboring the characteristics of a transcriptional activator leads to expression of lacZ, allowing for selection of relevant colonies. TFs during rice embryo development were isolated through this system. Approximately 200 confirmed positive colonies were obtained from screening 10(6) yeast colonies, and sequence analysis of conserved domains identified 75 independent cDNAs, 20 of which encoded plant TFs or co-activators, including members of the APETALA2 (AP2)/ethylene-responsive element-binding protein (EREBP), MYB and growth-regulating factor (GRF) families. Peptides encoded by 13 of the isolated cDNAs were classified as potential TFs or co-activators because of the presence of conserved TF-like domains. Additionally, 2, 11, and 13 clones encoded kinases, chromosome-related proteins, and unknown proteins, respectively, while the remaining 16 cDNAs were associated with specific functions seemingly unrelated to TFs. Expression pattern analysis of selected TF-encoding genes via RT-PCR revealed that these genes were expressed during seed development, with differential transcription observed during various stages. This work provides informative hints for further study of the regulatory mechanism of rice seed development and illustrates an identification strategy that will be of practical value for the isolation of TFs and co-activators associated with specific plant developmental processes.
摘要 开发了一种基于酵母的高效系统，用于分离编码转录因子（TFs）和具有转录激活功能的蛋白质（共激活因子）的植物cDNA。该系统由两个载体组成：（i）一个报告载体（pG221），其含有同工型1-细胞色素c（CYC1）核心启动子和β-半乳糖苷酶（lacZ）基因；（ii）一个cDNA文库构建载体（pYF503），它产生与GAL4结合域（GAL4-BD）融合的植物肽文库。具有转录激活剂特征的肽的表达导致lacZ的表达，从而可以选择相关菌落。通过该系统分离了水稻胚胎发育过程中的转录因子。从筛选的10⁶个酵母菌落中获得了约200个经确认的阳性菌落，保守结构域的序列分析鉴定出75个独立的cDNA，其中20个编码植物转录因子或共激活因子，包括APETALA2（AP2）/乙烯反应元件结合蛋白（EREBP）、MYB和生长调节因子（GRF）家族的成员。由于存在保守的类转录因子结构域，分离的13个cDNA编码的肽被归类为潜在的转录因子或共激活因子。此外，分别有2、11和13个克隆编码激酶、染色体相关蛋白和未知蛋白，而其余16个cDNA与似乎与转录因子无关的特定功能相关。通过RT-PCR对选定的编码转录因子的基因进行表达模式分析，结果表明这些基因在种子发育过程中表达，在不同阶段观察到差异转录。这项工作为进一步研究水稻种子发育的调控机制提供了有益的线索，并阐明了一种鉴定策略，该策略对于分离与特定植物发育过程相关的转录因子和共激活因子具有实际价值。
