基于转录组分析的杨树胚性愈伤组织形成的关键基因发掘及功能分析

Embryogenic callus of poplar is an ideal transgenic receptor material. Few reports focus on the key genes regulating embryogenic callus formation and their genetic effects. The poplar ‘shanxin’ (Populus davidiana×P. bolleana) with strong ability of embryogenic callus formation in our laboratory and the Nisqually-1, a Populus trichocarpa genotype which is not easy to form embryogenic callus, were used in our study. The microscopic morphology of embryogenic calli induced by poplar ‘shanxin’ and Nisqually-1, were observed by paraffin section and scanning electron microscopy. Differential expression of related genes during embryogenesis in different stages of formation was analyzed using transcriptional data and quantitative real-time PCR technique. Several key genes related embryogenic callus formation were explored. These key genes were cloned and their gene expression was analyzed. Then the key genes were introduced into the genome of poplar ‘shanxin’ and Nisqually-1 by Agrobacterium-mediated transformation and the genetic effects of transgenic plants and their progenies were studied. In this project, the molecular mechanism of embryogenic callus formation in poplar should be systematically analyzed from transcriptome level and the functions of key genes should be studied. This will promote the optimization of tissue culture characteristics and the improvement of transgenic efficiency in poplar.

杨树胚性愈伤组织是理想的转基因受体材料，对于调控胚性愈伤组织形成的关键基因及其遗传效应报道较少。本课题旨在采用本实验室胚性形成能力强的山新杨（Populus davidiana×P. bolleana）和胚性形成能力差的毛果杨（Populus trichocarpa, Nisqually-1）为受体基因型诱导出胚性愈伤组织，通过石蜡切片和扫描电镜观察胚性愈伤组织细胞的微观形态。利用胚性愈伤形成不同时期的转录组数据和实时定量PCR技术分析胚性发生过程中相关基因的表达差异，发掘几个与胚性愈伤形成相关的关键基因，克隆关键基因并对其进行表达分析，通过根癌农杆菌转化法将关键基因分别导入山新杨和毛果杨基因组中，并对转基因植株及其后代遗传效应进行研究。本课题从转录组水平系统探析杨树胚性愈伤形成的分子机理，研究胚性形成关键基因的基因功能，从而促进杨树组织培养特性的优化和杨树转基因效率的提高。

本项目采用胚性形成能力强的毛白杨‘BJHR01’ (Populus tomentosa，Pto)和山新杨(Populus davidiana × Populus bolleana，Pdb)，以及胚性形成能力差的毛果杨(Populus trichocarpa，Ptr)为受体基因型诱导出胚性愈伤组织，通过体式显微镜和扫描电镜观察胚性愈伤组织细胞的微观形态。对胚性愈伤组织形成不同时期的转录组数据进行了生物信息学分析，发现MAPK信号传导途径和植物激素信号转导途径参与植物激素合成代谢，与胚性愈伤组织形成有重要关联。通过qRT-PCR鉴定了这2个通路上差异基因的表达情况，最终确定了4个候选基因：分别为与细胞伸长相关的生长素响应因子ARF3和ARF5基因；与细胞分裂相关的B型反应调节因子ARR2基因；与乙烯调控途径相关的Ein3基因。在毛白杨‘BJHR01’克隆了这4个关键基因，构建了RNAi抑制表达和过量表达载体，转化毛白杨‘BJHR01’，分别获得了4个关键基因的RNAi抑制表达和过表达转基因株系，对转基因植株及其后代遗传效应进行研究。最终发现Ein3基因的过量表达降低了愈伤组织的形成率和分化率，同时Ein3基因的抑制表达显著促进了愈伤组织的形成和分化，结果表明Ein3基因对愈伤组织的形成和分化有一定的相关性。.本项目综合前人报道的文献资料，从转录组数据分析来确定杨树胚性愈伤形成的关键基因，研究关键基因的作用机制，从分子水平上探析杨树胚性发生的机理。这将有利于我们利用分子手段来改造现有的杨树育种手段，优化杨树组织培养特性，促进杨树转基因研究及种质资源的拓展。