Rop信号影响尾叶桉分化和广谱抗病性的机理研究

To promote callus differentiation and confer broad-spectrum disease resistance on Eucalyptus Urophylla, synthetic pathogen-inducible promotors (SPIPs) are designed to regulate SWAP70 expression. Rho-related GTPase of plants (Rop), a key GTPases involved in diverse signaling processes including immunity, growth and development, could be actived by interacting with SWAP70, which replaces bound GDP by GTP converting the molecular switch to the active conformation. .GWSF-mini35S promotor with low background transcriptional activity, screened from 8 SPIPs at previous study, could be induced by salicylic acid, Ralstonia solanacearum and the spores of Phytophthora capsici. At the basis of GWSF-mini35S promotor, the factors influencing the transcriptional character of SPIPs will be optimized, including cis-element types, arrangement, location, etc. And the ideal SPIP, which achieves some advantages, such as more induction factors, low basal activity, high expression activity and quick expression initiation, will be selected to subsequent study. Using the results of SPIP transcriptional character as foundation and consulting the transcriptional character of pathogen-inducible promoters reported at published literature, the mathematical model concerning the relationship between transcriptional character and the sequence of pathogen-inducible promoters would be established by genetic algorithm or simulated annealing algorithm. .The cDNA sequences of Arabidopsis SWAP70 gene and Eucalyptus SWAP70 are amplified from leaf cDNA, respectively. Then, the SWAP70 genes are used to construct plant inducible expression vector under the control of optimized SPIP. The genes are introduced into E. Urophylla by Agrobacterium tumefaciens mediated transformation. And the stable transformations are confirmed by PCR and Southern blot. .The interactions between SWAP70 and Ropx in callus differentiation and disease resistance of E. Urophylla are studied by yeast two-hybrid system. The cloned SWAP70 genes are used to construct bait voctor. The full-length Rop genes are isolated from Eucalyptus by PCR-RACE and they are used to construct prey voctors..In order to evaluate the effect on differentiation of adventitious buds of SWAP70 differential expression, it was induced by different inductor at callus differentiation stage of E. Urophylla. The content of hormone and reactive oxygen species (ROS) and expression changes of related enzymes are detected..Regulated SWAP70 expression with several pathogenic factors at growth stage of transgenic plant, the content of ROS, the expressional level of pathogenesis-related proteins and related enzymatic activities are detected. .The project is a new trials of tree broad-spectrum resistance by genetic engineering. It will offer helpful references to the design of SPIP and accumulate experiment evidence to clarify Rop signal network in Eucalyptus.

林木转基因抗病存在广谱抗性、持久抗性和低转化率等困难。设计人工病原物诱导启动子(SPIP)调控SWAP70表达，激活信号网络的关键节点Rop，促进尾叶桉芽分化并使其具广谱抗病性。以GWSF-mini35S为基础，优化元件类型、排列、间隔序列等，获得理想的SPIP，建立序列设计与SPIP转录特性关系的数学模型。克隆SWAP70基因，构建SPIP调控SWAP70表达的载体，转化尾叶桉。克隆Rop基因，通过酵母双杂交筛选与SWAP70互作、参与抗病反应或影响愈伤分化的Rop蛋白。分别在愈伤分化和植株生长阶段，通过信号分子类型和浓度的变化诱导SPIP，调控SWAP70的表达，影响Rop活性，检测激素含量、ROS含量、相关酶活性及基因的表达变化，探讨Rop与愈伤分化和抗病间的信号互作。本研究是林木转基因广谱抗病的新尝试，可为SPIP的设计提供参考，为阐明桉树Rop抗病和愈伤分化信号网络积累资料。

应用F-box、S-box、Gst1-box、W-box、V-box、设计了16个人工病原诱导启动子(SPIP)。通过农杆菌介导法得到转基因尾叶桉和拟南芥。检测SPIP在4种双子叶植物和4种单子叶植物中的本底和诱导转录特性。发现插入元件使得SPIP具有更多诱导因子；插入序列靠近mini35S时，本底活性升高，受诱导活性增强；插入序列远离mini35S时，对本底活性影响小，但增强了改良启动子对其他诱导因子的诱导活性。筛选出的GWVSF、WGVFS启动子具有本底表达低、诱导因子广、启动表达快、诱导效率高等优点，是较为理想的SPIP，可广泛用于植物遗传改良。.研究了愈伤组织分化的机理。发现PBU、BAP和DMTU协同作用，通过影响rboh和APX基因的表达，调节活性氧代谢，促进尾叶桉不定芽的分化。以下胚轴为材料建立了高效的尾叶桉再生体系。用携带SPIP-gus的农杆菌转化愈伤组织，经共培养、延迟筛选、抗性筛选得抗性芽。转基因不定芽中SPIP转录活性与瞬时表达检测结果一致，表现为V片段插在上游和中游对本底活性影响小，V片段插在下游会造成改良启动子本底活性显著升高。.项目组多次重复实验表明，SWAP70影响愈伤组织活性氧代谢，对愈伤组织的分化不利，表现为褐化增多，不利于愈伤组织分化出不定芽。故项目组用拟南芥研究SWAP70基因在植物抗病中的作用。GWSF调控EuSWAP70表达激活下游抗性基因表达显著增强转基因拟南芥灰霉病抗性，NPR1，CHIB，WRKY，PDF，MPK6等抗病基因表达增强。.项目筛选出GWVSF、WGVFS启动子、发现了启动子优化的规律；建立了桉树组织中启动子活性检测的瞬时表达方法体系。找到了适合桉树再生分化的脲类细胞分裂素PBU，建立了尾叶桉再生体系和遗传转化体系；获得了转SPIP-gus尾叶桉、转GWSF-NPR1尾叶桉。GWSF调控EuSWAP70表达可使抗性基因表达显著增强、增强抗性。项目研究成果为桉树分子育种奠定了良好基础。