Development of an efficient method for the isolation of factors involved in gene transcription during rice embryo development.
Development of an efficient method for the isolation of factors involved in gene transcription during rice embryo development.
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DOI：
10.1111/j.1365-313x.2004.02037.x
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发表时间：
2004-04
期刊：
影响因子：
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通讯作者：
R. Ye;Q. Yao;Zhi-hong Xu;Hong-Wei Xue
中科院分区：
文献类型：
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作者：
R. Ye;Q. Yao;Zhi-hong Xu;Hong-Wei Xue
开发了一种有效的基于酵母的系统，用于分离编码转录因子（TF）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）含有异-1-细胞色素c（CYC 1）核心启动子和β-半乳糖苷酶（lacZ）基因的报告载体（pG 221）;和（ii）cDNA文库构建载体（pYF 503），其产生与GAL 4结合结构域（GAL 4-BD）融合的植物肽文库。具有转录激活因子特征的肽的表达导致lacZ的表达，从而允许选择相关集落。利用该系统分离了水稻胚发育过程中的转录因子。从筛选10（6）个酵母菌落中获得约200个确认的阳性菌落，并且保守结构域的序列分析鉴定了75个独立的cDNA，其中20个编码植物TF或共活化剂，包括APETALA 2（AP 2）/乙烯响应元件结合蛋白（EREBP）、MYB和生长调节因子（GRF）家族的成员。由于存在保守的TF样结构域，由13个分离的cDNA编码的肽被分类为潜在的TF或共激活剂。此外，2，11，和13个克隆编码激酶，染色体相关的蛋白质，和未知的蛋白质，分别，而其余16个cDNA与特定的功能似乎无关的TF。通过RT-PCR对所选TF编码基因的表达模式分析显示，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。本研究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，对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用价值。
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.