基于cAMP-PKA途径研究茶碱影响核盘菌菌核形成的相关机制

Mechanisms of Theophylline on Sclerotial Formation Based on cAMP-PKA Pathway

【目的】菌核在核盘菌生活史中占据核心地位,茶碱可作用于cAMP-PKA途径中的PDEs而影响生物体生长发育,本研究通过外源添加茶碱,研究茶碱对于核盘菌菌核的产生及其通路中相关因子的影响,其结果可为新型杀菌剂的开发提供参考。【方法】首先,通过外源添加不同浓度的茶碱,检测其对核盘菌菌丝生长抑制率及菌核形成情况的影响,其次,通过试剂盒检测茶碱对核盘菌菌丝体内cAMP含量的影响,最后,通过qRT-PCR方法检测外源添加茶碱对于核盘菌cAMP-PKA通路中相关因子的变化。【结果】外源添加终浓度为1~2.5 mmol/L的茶碱,对于核盘菌菌丝生长速率和菌核的形成均有抑制作用,而茶碱终浓度为0.5 mmoI/L对菌丝生长速率几乎没有影响。外源添加的茶碱主要是在菌核原基形成的S4期及菌核成熟的S5阶段提升胞内cAMP含量,而在S1~S3阶段和对照组胞内cAMP含量无差异。茶碱通过抑制PDE2的表达水平使得胞内cAMP含量升高,而下游基因PKA和RAS的表达量也相应减少,外源添加茶碱后ATG8的表达量显著上升。【结论】一定浓度的茶碱对于核盘菌菌丝生长速率和菌核的产生均有抑制作用,外源添加茶碱可通过抑制PDE2的表达而提高胞内cAMP含量,使得下游基因表达量相应减少。

Sclerotia play a core role in the life cycle of Sclerotinia sclerotiorum.%Theophylline can act on PDEs in the cAMP-PKA pathway and affect the growth and development of organisms.This study investigates the effects of exogenous theophylline on the production of sclerotia and related factors in the pathway,with the aim of providing a reference for the development of new fungicides.Different concentrations of theophylline were added externally to evaluate its effect on the growth inhibition rate and sclerotial formation of mycelium,and then the effect of theophylline on the cAMP content in mycelium was investigated using a reagent kit.Finally,the changes in relevant factors in the cAMP-PKA pathway were detected by qRT-PCR.The results showed that the addition of exogenous theophylline at a final concentration of 1 mmol/L to 2.5 mmol/L inhibited the growth rate of hyphae and the formation of sclerotia,while theophylline at a final concentration of 0.5 mmol/L had almost no effect on the growth rate of hyphae.The exogenous addition of theophylline mainly enhanced the intracellular cAMP content during the S4 stage of sclerotia primordia formation and the S5 stage of sclerotia maturation,while there was no difference in the intracellular cAMP content between the S1~S3 stages and the control group.Theophylline increased the intracellular cAMP content by inhibiting the expression level of PDE2,while the expression levels of the downstream genes PKA and RAS also decreased accordingly.The expression level of ATG8 significantly increased after the exogenous addition of theophylline.In conclusion,a certain concentration of theophylline has an inhibitory effect on the growth rate of Sclerotinia sclerotiorum hyphae and the production of sclerotia.Exogenous addition of theophylline can increase the intracellular cAMP content by inhibiting the expression of PDE2,resulting in a corresponding decrease in downstream gene expression.