【作者】 倪杨；

【导师】 梁军；

【作者基本信息】 中国林业科学研究院 ， 森林保护学， 2014， 博士

【摘要】 本研究以毛白杨（Populus tomentosa Carr.）和外生菌根（Ectomycorrhizae，ECM）真菌-美味牛肝菌（Boletus edulis Bull. ex Fr.）为试验材料，建立水培互作试验体系，针对菌-根之间在预共生阶段的信号分泌、识别及传导，收集含有水溶性信号因子的信号液，检测其对杨树根系先天免疫机制的诱导以及第二信使跨膜转运的触发，重点分析Ca2+和H2O2的跨膜转运与防御相关基因之间的级联关系，探讨ECM预共生阶段寄主植物早期免疫机制的启动模式。主要研究结果如下：1、设计两种模拟ECM早期共生阶段进行信号分泌、传导与识别的水培互作试验体系，分析其对候选共生相关基因表达的影响。在真菌侵染模拟体系中，根系中SOD, POD,PAL, APX和CIPK25种候选共生相关基因的表达水平在5d内呈显著上调趋势，说明ECM真菌对寄主根系的直接接触，能够快速诱导这两类基因的转录表达。在信号传导模拟体系中，根系SOD, POD, PAL和APX4种基因的表达呈缓慢上升趋势，与真菌侵染模拟体系中的表达趋势一致，除APX之外，其它3个基因的转录水平在互作过程中均显著上调，并且SOD, POD和APX的mRNA含量在互作第3d达到最高值；CIPK2基因在互作过程中呈显著上调表达，互作1d后该基因的转录水平即可达到一个小高峰，并且显著高于对照和真菌侵染模拟体系，说明该体系下的ECM真菌能够分泌水溶性信号因子，有效地诱导寄主共生相关基因上调表达，初步验证了信号传导模拟体系对研究ECM早期信号事件的可行性和有效性，并且该体系下的ECM真菌，在互作第3d所分泌的信号因子能够有效诱导寄主共生相关基因的上调表达。2、在信号传导模拟体系的基础上，收集、制备菌-根共生信号液以及毛白杨根系分泌物、真菌分泌物的水溶液，对比分析其对根系SOD、POD和CIPK2基因转录水平的影响。结果表明，根系分泌物对基因的上调表达没有诱导作用，而真菌分泌物则能够显著诱导这些基因表达的上调，并且均在互作12h达到最高值。信号液对寄主共生相关基因转录水平的上调诱导作用最为显著，其中对CIPK2基因表达上调的诱导最为迅速，分别于3h和9h时达到极显著水平，呈“双高峰”的形式保持基因表达的上调趋势；SOD基因的转录水平于6h达到一个小高峰，并于12h达到最高值；而POD基因的表达量在整个互作期间均处于上调状态，并且上调幅度大，变化显著。通过对比分析认为，在正常生长状态下，ECM真菌的分泌物能够诱导寄主根系防御基因的表达，但在菌-根互相体系中，双方通过信号因子的交流而感知彼此的存在后，进一步分泌水溶性共生信号并将其传递，此时，寄主感知真菌源共生信号后，以启动自身免疫机制的方式作出回应，从而提高防御基因的表达。3、利用非损伤扫描离子选择性电极技术（Scanning Ion-selective Electrode Technique，SIET）研究共生信号诱导下根系的Ca2+跨膜转运的动态变化模式。根系伸长区的Ca2+流在稳定环境下处于小内流状态，当用信号液瞬时处理根系后，Ca2+首先呈现出巨大的外流状态，但在随后的30min内，外流逐渐减弱，并稳定于小外流状态。伴随着信号因子与根系之间的识别与诱导，Ca2+的跨膜转运流向逐渐转变为稳定的内流状态，并且流速随时间逐渐增大，而对照则逐渐回归平稳的小内流状态。此外，根系在真菌分泌物和根系分泌物的瞬时处理下，Ca2+的跨膜转运形式与信号液处理的一致，而后期则逐渐恢复到与对照一样的小内流状态，说明共生信号液里含有某种信号分子诱导根系表皮细胞的Ca2+跨膜内流，或者是植物本身在识别了真菌源共生信号后，将Ca2+作为第二信使，通过自身的调控使其处于内流状态，从而将真菌源共生信号在体内进行传递。4、利用非损伤扫描极谱电极技术（Scanning Polarographic Electrode Technique，SPET）研究信号因子诱导下根系释放H2O2的动态模式。根系伸长区的H2O2在平衡条件下表现为内吸外排的平衡状态，在不同的瞬时处理下，根系起初释放大量的H2O2，但在最后均逐渐趋向于小吸收状态。随着信号因子的诱导，根系释放H2O2的水平也在不断提高，并在互作9h时达到最大。此外，真菌分泌物也能在24h内诱导根系产生H2O2，而根系分泌物的处理则总体上抑制了H2O2的释放，仅在3h处于较低的释放水平，然后一直表现为平衡或内吸状态。本试验首次展示了根系受ECM信号因子的诱导产生H2O2的动态模式。5、通过药理学实验分析H2O2和Ca2信号与防御反应之间的关系，探讨ECM免疫机制的启动模式。药理学试验证明，LaCl3（质膜钙离子通道抑制剂）能够有效抑制寄主根系Ca2+外流以及H2O2的产生，使Ca2+和H2O2一直处于内流状态。而DPI（质膜NADPH氧化酶抑制剂）只能有效抑制寄主根系H2O2的产生，但对Ca2+跨膜转运的动态模式没有影响，只是在3h时降低了Ca2+跨膜外流的速度。此外，LaCl3处理寄主根系后，能够显著抑制CIPK2、SOD和POD基因的转录水平，而DPI的处理只抑制了SOD和POD基因的转录水平，并不影响CIPK2基因的正常表达。因此推测寄主免疫机制的启动模式表现为，毛白杨在ECM预共生阶段，信号因子诱导寄主根系Ca2+跨膜内流，其与CIPK2基因结合后，调控根系产生大量的H2O2，以此诱导防御基因SOD和POD基因的转录表达。更多还原

【Abstract】 Establishment of popla（rPopulus tomentosa）and ectomycorrhizal fung（iBoletus edulis）hydroponic interaction experiment system and collection the signal liquid containingwater-soluble signal factors to detect the innate immunity response characteristics and secondmessenger transmembrane transport of host plant roots during the presymbiotic stage. Focusingon analysis of cascade relationship between Ca2+and hydrogen peroxide(H2O2) transmembranetransport and symbiosis related genes, discussion the startup mode about host plants earlyimmune mechanism during the presymbiotic stage of ECM. The main results and conclusionsare as follow:1. Design two kinds of hydroponic interaction experiment system to simulate signalsconduction events between fungi and plant in the early stage of ECM symbiosis, analysis theeffect of symbiotic signals on the candidate symbiosis-related genes expression. In thesimulation system of ECM fungi infection in host plant roots, the expression profiles of SOD,POD, PAL, APX and CIPK2showed significantly rising trend in5days, indicating that ECMfungi direct contact on host roots, will be quickly induced the transcriptional expression ofthese genes. While in the simulation system of signals transduction, genes expression patternsof SOD, POD, PAL and APX showed slowly rising state in plant roots, but these trends wereconsistent with the fungi infection system, in addition to APX, the transcription levels of otherthree genes all showed significantly increased during the interaction process, and the mRNAcontent of SOD, POD and APX reached the highest value in the interaction of the third day.The expression profile of CIPK2kept a significantly up-regulated state in the interactionprocess, which reached the peak after the first day and significantly higher than the control andthe simulation system of fungi infection, proved that ECM fungi effectively induced theexpression of symbiotic related genes in plant roots through secreted water-soluble signalfactors, and preliminary verified the effectiveness of signaling simulation system to studysymbiosis signaling events during the ECM prosymbiotic stage. In addition, through a comprehensive comparative analysis, on the third day after interaction, considered that thesymbiotic signals secreted by ECM fungi can effectively induce symbiosis-related genesexpression for plant roots in this system.2. Preparated and collected symbiosis signal liquid based on signal transductionsimulation system, as well as collected aqueous solutions of roots exudates and fungi exudates,contrasted analysis the impact on symbiosis-related genes expression in these treatment. Rootexudates have no significant effect on the expression of SOD、POD and CIPK2, but theexpression of these genes induced by fungi exudates showed significantly increased andreached the highest value in the interaction of12h. Symbiotic signals have the mostsignificantly induction for root genes expression, and which induced the most rapidly changesin transcription level of CIPK2, respectively significantly induced up-regulated expression in3h and9h to form a “double peak” showed continued upregulated. SOD reached two peaks inthe6h and12h, respectively, and its transcription level in12h achieved the highest value, PODup-regulated expression of the whole process showed significantly and rapidly trend. Throughthe contrast analysis that up-regulated expression of the roots immune genes can be induced byECM fungi secretion, but in the recognition process, fungi or roots through communicationsignal factors to sense the presence of each other, and further secret of soluble signalingmolecules to expand pre-symbiotic interaction phase of signal transduction, at this time, hostplant roots will improve the expression of immune response genes and initiative their earlyimmune mechanisms after perceived fungi source signaling molecules.3. By means of scanning ion-selective electrode technique to research dynamic changes ofCa2+transmembrane transport in root organizations induced by symbiotic signal factors. TheCa2+flow on root elongation zone showed a small inflow state in a stable environment, but thehuge outflow occured when was instantaneous treated with signals solution, and graduallystabilized at a small outflow of state. With roots were induced by signal factors, Ca2+transmembrane transport flows gradually transformed into a stable inflow state, and the flowrate increased with time, while the control is gradually returned to a steady small inflow state.In addition, under the instantaneous treated of fungi secretions and root secretions, the formation of Ca2+transmembrane transport was consistent with signals solution, while thelatter was gradually restored to the small inflow state like control, suggested that the symbioticsignals solution contained some kinds of signal molecules which induced Ca2+internal flowacross the membrane in roots epidermal cells, or taked the Ca2+as second messenger toregulate Ca2+influx after identified fungi signals, finally transducted the symbiotic relatedsignals in the plant body.4. Researched the dynamic changes of H2O2production mode in root organizationsinduced by symbiotic signal factors through scanning ion-selective electrode technique. H2O2showed a stable state in equilibrium conditions, while the root released large amounts of H2O2under different transient treatments, and gradually moving to small absorbed state at last. Withthe signal factors inducted, the level of it released H2O2were also rising and reached themaximum when interacted at9hours. Fungi secretions induced root produced H2O2within24hours, but root secretions inhibited the release on the whole which showed the balance orabsorbed state except released at a very lowly level in3hours. This experiment firstlydemonstrated for the dynamic mode of H2O2generated induced by signal factors in plant roots.5. By means of drug pharmacology experiments studied the action of H2O2and Ca2+signals in the early immune response, and explored the initiation mode of immune in ECMpresymbiotic stage. Pharmacological experiments demonstrated that the Ca2+outflow and H2O2production were effectively inhibited by LaCl3(plasma membrane calcium channel inhibitor),effectively inhibited Ca2+efflux host roots and H2O2generation, so that the root tissues havebeen in a absorption state for Ca2+and H2O2. While the DPI (plasma membrane NADPHoxidase inhibitors) could only effectively inhibited the production of H2O2in host roots, didnot affect the dynamic mode for Ca2+transmembrane transport, only reduced the speed of Ca2+outflows across membrane in3hours. In terms of gene expression profiles, the transcriptionlevel of CIPK2, SOD and POD genes were significantly inhibited when host plant roots betreatmented by LaCl3. But by means of DPI only inhibited the transcription levels of SOD andPOD genes, did not affect the normal expression of CIPK2genes. Thus speculated that signalfactors induced the Ca2+inflowed host roots across membrane during the prosymbiotic stage, and combined with CIPK2gene to regulate roots produced a large number of H2O2, in order toinduce transcriptional expression of immune related genes and initiative the host plants earlyimmune mechanisms.更多还原