美味牛肝菌-毛白杨根系水培互作体系的构建以及抗逆相关基因对信号因子的响应
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摘要
本研究以毛白杨(Populus tomentosa)和外生菌根(ectomycorrhizae, ECM)真菌——美味牛肝菌(Boletus edulis)为试验材料,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。在信号传导模拟体系中,根系4种基因(SOD、POD、PAL和APX)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,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。在信号传导模拟体系的基础上,分析菌-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根系SOD、POD和CIPK2基因转录水平的影响。结果表明,信号液对CIPK2基因表达上调的诱导最为迅速,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12 h时呈现单峰;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。通过对比分析推测,在菌-根互作体系中,双方通过信号因子的交流而感知彼此的存在后,分泌并传递水溶性共生信号,寄主感知真菌源共生信号后启动自身免疫机制,提高防御基因的表达。
In this study, Populus tomentosa and ectomycorrhizae(ECM) Boletus edulis were used as experimental materials to design two systems. In the signal transduction simulation system, the expression of four genes(SOD, POD, PAL and APX) in roots showed a slow upward trend, which was consistent with the expression trend in the fungal infection simulation system, indicating that the ECM fungi in this system could secrete water-soluble signal factors and effectively induce host symbiosis. On the basis of the signal transduction simulation system, the fungal-root symbiotic signal solution and the aqueous solution of root exudates and fungal secretions of P. tomentosa were collected and compared, and the effects on the transcription levels of SOD, POD and CIPK2 genes in roots were compared and analyzed. The up-regulation of CIPK2 expression was the most rapid, and the expression of gene expression was up-regulated as a "double peaks" mode. The transcription of SOD gene was expressed after 12 hours. While the expression level of POD gene was up-regulated during the whole interaction period, and the up-regulation range was significant. Through comparative analysis, it is believed that in the mutual system of fungal-roots, the two sides perceive each other through the communication of signal factors, secrete and transmit water-soluble symbiotic signals. Then, the host senses the symbiotic signal of the fungal source to initiate the autoimmune mechanism and improve the expression of defense gene.
In this study, Populus tomentosa and ectomycorrhizae(ECM) Boletus edulis were used as experimental materials to design two systems. In the signal transduction simulation system, the expression of four genes(SOD, POD, PAL and APX) in roots showed a slow upward trend, which was consistent with the expression trend in the fungal infection simulation system, indicating that the ECM fungi in this system could secrete water-soluble signal factors and effectively induce host symbiosis. On the basis of the signal transduction simulation system, the fungal-root symbiotic signal solution and the aqueous solution of root exudates and fungal secretions of P. tomentosa were collected and compared, and the effects on the transcription levels of SOD, POD and CIPK2 genes in roots were compared and analyzed. The up-regulation of CIPK2 expression was the most rapid, and the expression of gene expression was up-regulated as a "double peaks" mode. The transcription of SOD gene was expressed after 12 hours. While the expression level of POD gene was up-regulated during the whole interaction period, and the up-regulation range was significant. Through comparative analysis, it is believed that in the mutual system of fungal-roots, the two sides perceive each other through the communication of signal factors, secrete and transmit water-soluble symbiotic signals. Then, the host senses the symbiotic signal of the fungal source to initiate the autoimmune mechanism and improve the expression of defense gene.
引文
Baptista P,Martins A,Pais MS,et al(2007).Involvement of reactive oxygen species during early stages of ectomycorrhiza establishment between Castanea sativa and Pisolithus tinctorius.Mycorrhiza,17(3):185-193
Brundrett MC,Cairney JW(2002).Ectomycorrhizas in Plant Communities.In:Sivasithamparam K,Dixon KW,Barrett RL(eds).Microorganisms in Plant Conservation and Biodiversity.Netherland:Kluwer Academic Publishers,105-150
Brunner I,Amiet R,Zollinger M,et al(1992).Ectomycorrhizal syntheses with Picea abies and three fungal species:a case study on the use of an in vitro technique to identity naturally occurring ectomycorrhizae.Mycorrhiza,2:89-96
Cheng YE(2010).Ectomycorrhizal fungi enchance drought resistance of Populus ussuriensis(dissertation).Yangling:Northwest Agriculture and Forestry University(in Chinese with English abstract)[程玉娥(2010).外生菌根真菌提高杨树抗旱性的研究(学位论文).杨凌:西北农林科技大学]
Hebe G,Hager A,Salzer P(1999).Initial signalling processes induced by elicitors of ectomycorrhiza-forming fungi in spruce cells can also be triggered by G-protein-activating mastoparan and protein phosphatase-inhibiting cantharidin.Planta,207(3):418-425
Martin F,Kohler A,Duplessis S(2007).Living in harmony in the wood underground:ectomycorrhizal genomics.Curr Opin Plant Biol,10(2):204-210
Mu QL(2013).The influence of inoculation of arbucular mycorrhizal fungi and ectomycorrhizal fungi on Populus cana(dissertation).Huhhot:Inner Mongolia University(in Chinese with English abstract)[牧骑勒(2013).丛枝菌根真菌与外菌根真菌单接种和混合接种对白皮杨苗木生长的影响(学位论文).呼和浩特:内蒙古大学]
Ni Y(2014).Ectomycorrhiza fungi initiate the early immune mechanism in Populus tomentosa roots during the presymbiotic phase(dissertation).Beijing:Chinese academy of forestry(in Chinese with English abstract)[倪杨(2014).毛白杨外生菌根预共生阶段的免疫机制启动模式(学位论文).北京:中国林业科学研究院]
Ostonen I,L?hmus K(2003).Proportion of fungal mantle,cortex and stele of ectomycorrhizas in Picea abies(L.)Karst.in different soils and site conditions.Plant Soil,257(2):435-442
Salzer P,Hebe G,Hager A(1997).Cleavage of chitinous elicitors from the ectomycorrhizal fungus Hebeloma crustuliniforme by host chitinases prevents induction of K+and Cl-release,extracellular alkalinization and H2O2synthesis of Picea abies cells.Planta,203(4):470-479
Schwacke R,Hager A(1992).Fungal elicitors induce a transient release of active oxygen species from cultured spruce cells that is dependent on Ca2+and protein-kinase activity.Planta,187(1):136-141
Tang MJ,Guo SX(2000).Research advances on mechanism of mycorrhiza enhancement in plant disease resistance.Microbiology,27(6):446-449(in Chinese with English abstract)[,(2000).Microbiology,27(6):446-449(in Chinese with English abstract)[唐明娟,郭顺星(2000).菌根增强植物抗病性机理的研究进展.微生物学通报,27(6):446-449]
Trappe JM(1977).Selection of fungi for ectomycorrhizal inoculation in nurseries.Annu Rev Phytopathol,15:203-222
Vralstad T,Schumacher T,Taylor AFS(2002).Mycorrhizal synthesis between fungal strains of the Hymenoscyphus ericae aggregate and potential ectomycorrhizal and ericoid hosts.New Phytol,153(1):143-152
Yang L,Zhou GY,Liang J(2016).Antifungal mechanism of two biocontrol strains on poplar canker.Plant Prot,42(2):47-54(in Chinese with English abstract)[杨蕾,周国英,梁军(2016).杨树溃疡病生防菌株的抑菌机理研究.植物保护,42(2):47-54]
Yuan ZL,Chen LQ(2007).The mechanism of signal recognition and transduction in the establishment of mycorrhizal associations.Microbiology,34(1):161-164(in Chinese with English abstract)[袁志林,陈连庆(2007).菌根共生体形成过程中的信号识别与转导机制.微生物学通报,34(1):161-164]
Brundrett MC,Cairney JW(2002).Ectomycorrhizas in Plant Communities.In:Sivasithamparam K,Dixon KW,Barrett RL(eds).Microorganisms in Plant Conservation and Biodiversity.Netherland:Kluwer Academic Publishers,105-150
Brunner I,Amiet R,Zollinger M,et al(1992).Ectomycorrhizal syntheses with Picea abies and three fungal species:a case study on the use of an in vitro technique to identity naturally occurring ectomycorrhizae.Mycorrhiza,2:89-96
Cheng YE(2010).Ectomycorrhizal fungi enchance drought resistance of Populus ussuriensis(dissertation).Yangling:Northwest Agriculture and Forestry University(in Chinese with English abstract)[程玉娥(2010).外生菌根真菌提高杨树抗旱性的研究(学位论文).杨凌:西北农林科技大学]
Hebe G,Hager A,Salzer P(1999).Initial signalling processes induced by elicitors of ectomycorrhiza-forming fungi in spruce cells can also be triggered by G-protein-activating mastoparan and protein phosphatase-inhibiting cantharidin.Planta,207(3):418-425
Martin F,Kohler A,Duplessis S(2007).Living in harmony in the wood underground:ectomycorrhizal genomics.Curr Opin Plant Biol,10(2):204-210
Mu QL(2013).The influence of inoculation of arbucular mycorrhizal fungi and ectomycorrhizal fungi on Populus cana(dissertation).Huhhot:Inner Mongolia University(in Chinese with English abstract)[牧骑勒(2013).丛枝菌根真菌与外菌根真菌单接种和混合接种对白皮杨苗木生长的影响(学位论文).呼和浩特:内蒙古大学]
Ni Y(2014).Ectomycorrhiza fungi initiate the early immune mechanism in Populus tomentosa roots during the presymbiotic phase(dissertation).Beijing:Chinese academy of forestry(in Chinese with English abstract)[倪杨(2014).毛白杨外生菌根预共生阶段的免疫机制启动模式(学位论文).北京:中国林业科学研究院]
Ostonen I,L?hmus K(2003).Proportion of fungal mantle,cortex and stele of ectomycorrhizas in Picea abies(L.)Karst.in different soils and site conditions.Plant Soil,257(2):435-442
Salzer P,Hebe G,Hager A(1997).Cleavage of chitinous elicitors from the ectomycorrhizal fungus Hebeloma crustuliniforme by host chitinases prevents induction of K+and Cl-release,extracellular alkalinization and H2O2synthesis of Picea abies cells.Planta,203(4):470-479
Schwacke R,Hager A(1992).Fungal elicitors induce a transient release of active oxygen species from cultured spruce cells that is dependent on Ca2+and protein-kinase activity.Planta,187(1):136-141
Tang MJ,Guo SX(2000).Research advances on mechanism of mycorrhiza enhancement in plant disease resistance.Microbiology,27(6):446-449(in Chinese with English abstract)[,(2000).Microbiology,27(6):446-449(in Chinese with English abstract)[唐明娟,郭顺星(2000).菌根增强植物抗病性机理的研究进展.微生物学通报,27(6):446-449]
Trappe JM(1977).Selection of fungi for ectomycorrhizal inoculation in nurseries.Annu Rev Phytopathol,15:203-222
Vralstad T,Schumacher T,Taylor AFS(2002).Mycorrhizal synthesis between fungal strains of the Hymenoscyphus ericae aggregate and potential ectomycorrhizal and ericoid hosts.New Phytol,153(1):143-152
Yang L,Zhou GY,Liang J(2016).Antifungal mechanism of two biocontrol strains on poplar canker.Plant Prot,42(2):47-54(in Chinese with English abstract)[杨蕾,周国英,梁军(2016).杨树溃疡病生防菌株的抑菌机理研究.植物保护,42(2):47-54]
Yuan ZL,Chen LQ(2007).The mechanism of signal recognition and transduction in the establishment of mycorrhizal associations.Microbiology,34(1):161-164(in Chinese with English abstract)[袁志林,陈连庆(2007).菌根共生体形成过程中的信号识别与转导机制.微生物学通报,34(1):161-164]