杨树转录延伸复合体Elp基因参与干旱耐受的分子机制

Populus is an important woody plant, it is also a model plant used for studying cell morphology, gene expression and metabolic regulation during wood formation. Drought is a major abiotic stress negatively affecting wood production. Osmolates, transcription factors and plant hormones are involved in plant abiotic stress response, especially there is a tight relationship between ABA and drought stress response..Elongator complex is a highly conserved complex consisting of six protein subunits (Elp1-Elp6). Certain subunits participate in the modification of ncm5U (5-carbamoyl- methyl-uridine) nucleoside of tRNA. Arabidopsis elp1 mutant showed increased drought tolerance than wild type, stomatal closure hypersensitivity to ABA, and elevated oxidative stress tolerance. It is suggested there is functional conversation of plant Elp proteins and yeast Elp proteins. There are multiple Elp genes involved in regulation of vegetative growth, drought response and ncm5U nucleoside modification in Arabidopsis..This project will first identify Populus Elp genes by homology comparison to the Elp genes in yeast and Arabidopsis. By constructing over-expression and RNAi transgenic populus, we aim to establish the relevance analysis between Populus Elp genes, ABA response, drought stress tolerance and ncm5U modification. By yeast two-hybrid analysis, we aim to identify new members associated with Populus elongator complex, underling the role of each Elp proteins in ABA response, drought tolerance and ncm5U nucleoside modification, and the physical interaction between them. Some drought-tolerant Elp transgenic populus trees will be obtained.

杨树是研究树木生长发育，特别是木材形成的模式植物。干旱是影响木材产量的一种主要非生物胁迫，其中脱落酸（ABA）在植物干旱胁迫响应中扮演重要角色。.转录延伸复合体由六个蛋白质（Elp1-6）组成，功能高度保守，参与mRNA转录延伸和tRNA特异核苷ncm5U的修饰。植物Elp基因参与营养生长和干旱胁迫。这是植物学新兴的研究内容。.本项目延续前期在拟南芥中已有研究，（1）分离毛果杨Elp同源基因，研究其非生物胁迫应答、组织特异性和蛋白亚细胞定位；（2）通过酵母双杂交预测杨树转运延伸复合体的组成以及成员间的相互作用；（3）使用Elp超量表达和RNAi的杨树转基因植株测定它们的干旱耐受力、ABA敏感性、ncm5U核苷修饰和基因表达谱，分析杨树Elp表达、ncm5U核苷修饰、ABA响应和干旱耐受力之间的相关性，解析Elp基因影响ncm5U核苷修饰和干旱耐受的分子机制；（4）获得抗旱的转基因杨树。

本项目在题为“杨树转录延伸复合体ELP基因参与干旱耐受的分子机制”的一年期初期研究中，构建了杨树ELP复合体成员编码基因的超量表达和RNAi干扰载体，进行了农杆菌介导的杨树转化并获得了部分RNAi干扰的阳性株系。根据候选基因的启动子序列构建以GFP-GUS为报道基因的基因时空特异表达载体；根据候选基因的编码区构建超量表达载体。另外，根据不同杂交杨品系的生长特性，在2013年从天津大学资源与环境学院及北京林业科学研究院分别新引入了2个可用于杨树转化的杂交杨品系：（1）717杨-Populus tremula x P. alba (717-1B4 genotype)；（2）84k杨-P. alba×P. glandulosa) 。这两种杨树在离体叶片愈伤分化和组培苗的生长速度上有一定的优势。在杨树转化体系上采用717杨，在不同阶段梯度有针对性变更培养基中的激素组分和浓度，对抗性芽的分化和生根采取不同策略，已经得到部分候选基因RNAi干扰的阳性芽（表1）。所有ELP基因的超量表达和启动子活性分析正在进行中，其中超量表达采用毛果杨P.trichocarpa的cDNA作为模板，进行杨树ELP1-6同源基因的超表达转基因载体的构建，杨树转化受体为717杨。经过叶盘转化法，通过愈伤诱导，抗性芽的分化和生根等步骤，最终得到每个基因的超量表达转基因株系，通过PCR和RT-PCR鉴定阳性苗的抗性和目标基因的表达，再通过HPLC分析修饰核苷的变化，通过干旱处理和ABA外源施加分析转基因植株与对照在抗逆性和ABA响应上的表型差异。