水杨酸和茉莉酸/乙烯信号通路关键基因在月季-黑斑病菌互作中的表达模式

Expression Patterns of Key Genes of Salicylic Acid and Jasmonic Acid/Ethylene Signaling Pathways in the Interaction between Rose and Diplocarpon rosae

【目的】监测月季水杨酸和茉莉酸信号途径关键基因的表达动态,以阐明水杨酸、茉莉酸/乙烯抗病信号途径在月季响应黑斑病菌过程中调控机制。【方法】以与黑斑病菌(cfcc87205)呈亲和互作的月季‘粉和平’离体叶片为材料,设置无菌水诱导2 h接种(CK+IN)、2 mmol·L-1SA诱导2 h不接种(SA+NO)、2 mmol·L-1SA诱导2 h接种(SA+IN)、0.2 mmol·L-1MeJA诱导2 h不接种(JA+NO)和0.2 mmol·L-1MeJA诱导2 h接种(JA+IN)5个处理和空白对照组(CK),在接种后16~144 h取样并通过RT-qPCR技术监测水杨酸信号途径ICS、NPR1、PR1和茉莉酸信号途径相关基因AOS、JAR1、COI1、MYC2共7个基因的表达动态,对不同处理下各基因在同一时间点的表达量差异和同一处理下各基因的表达动态进行综合分析。【结果】1)CK+IN处理中,ICS下调表达,NPR1在16 h上调、在48~144 h持续下调表达(P<0.05),PR1和JAR1除分别在48和16 h下调表达外其余时间点与CK处理无显著差异(P<0.05),AOS在16 h上调表达、72 h下调表达、在24、48和144 h与CK处理无显著差异(P<0.05),COI1在24~72 h上调表达(P<0.05),MYC2在16 h上调表达、在48 h与CK处理无显著差异、在24、72和144 h下调表达(P<0.05);2)SA+IN处理中,ICS、NPR1、PR1和AOS上调表达(P<0.05),JAR1与CK+IN处理中无显著差异(P<0.05),COI1下调表达(P<0.05),MYC2除在接种16和48 h与CK+IN处理中无显著差异外其余时间点上调表达(P<0.05);3)JA+IN处理中,ICS在16 h下调表达、在24~144 h上调表达(P<0.05),PR1在16、72和144 h上调表达、在24和48 h与CK+IN处理无显著差异(P<0.05),AOS和MYC2上调表达且在48 h前上调幅度较大(P<0.05),JAR1除在24 h上调表达外、其余时间点与其在CK+IN处理中无显著差异(P<0.05),NPR1和COI1变化趋势同SA+IN处理。结果表明,病原菌对水杨酸和活性茉莉酸的合成有抑制作用,SA+IN处理可提高水杨酸信号途径相关基因的表达、促进茉莉酸的生成、但对活性茉莉酸的合成影响不大,接种48 h前SA+IN处理可削弱病原菌对MYC2的诱导效应,接种48 h后SA+IN处理在提高ICS和PR1表达的同时也可提高MYC2的表达;JA+IN处理在促进活性茉莉酸合成和PR1表达的同时,也提高MYC2的表达。4)在SA+NO和JA+NO处理中,7个基因的表达量相对于CK不同程度的发生改变,但其变化趋势与相应的SA+IN和JA+IN处理不一致,说明病原菌影响外源水杨酸和茉莉酸对寄主相应信号通路基因的诱导表达。【结论】黑斑病菌对月季的水杨酸和茉莉酸/乙烯信号途径有抑制作用,且在接种48 h前主要抑制水杨酸信号途径、在接种48 h后主要抑制茉莉酸/乙烯信号途径。病原菌可能通过操纵月季MYC2的上调表达来实现其对水杨酸和茉莉酸/乙烯信号途径的双重抑制。

【Objective】In this paper,the expression dynamics of key genes in salicylic acid and jasmonic acid/ethylene signaling pathway in rosa were monitored in order to clarify the roles and regulatory mechanisms of salicylic acid and jasmonic acid/ethylene signaling pathways in rose in response to Diplocarpon rosae.【Method】The detached leaves of cultivar Rosa‘MEIbil’compatible with D.rosae(cfcc87205)was used as the test materials,and five treatments,including inoculation with D.rosae(CK+IN),2 mmol·L-1 SA-induced for 2 h and non-inoculation(SA+NO),2 mmol·L-1 SA-induced for 2 h and inoculation(SA+IN),0.2 mmol·L-1MeJA-induced for 2 h and non-inoculation(JA+NO),0.2 mmol·L-1MeJA-induced for 2 h and inoculation(JA+IN),and blank control(CK),were set.The leaves were sampled from 16-144 h post-treatments and the expressions of ICS,NPR1 and PR1 in salicylic acid signaling pathway and the expressions of AOS,JAR,COI1 and MYC2 related with jasmonic acid signaling pathway were detected through RT-qPCR analysis technique.The difference of expression amount of each gene at the same time point over different treatments and the expression dynamics of each gene under the same treatments were analyzed comprehensively.【Result】In CK+IN treatment,ICS was down-regulated(P≤0.05),NPR1 was up-regulated at 16 h and down-regulated continuously from 48-144 h post-inoculated(P≤0.05),PR1 and JAR1 were down-regulated at 48 and 16 h post-inoculated(P≤0.05),respectively,but there were no significant differences with CK in other time points.AOS was up-regulated at 16 h,down-regulated at 72 h,and there was no significant difference with CK treatment at 24,48 and 144 h post-inoculated(P≤0.05).COI1 was up-regulated from 24 to 72 h post-inoculated(P≤0.05).MYC2 was up-regulated at 16 h,and down-regulated at 24,72 and 144 h post-inoculated(P≤0.05),however there was no significant difference with CK treatment at 48 h.In SA+IN treatment,ICS,NPR1,PR1 and AOS were up-regulated(P≤0.05),there was no significant difference in JAR1 expression between SA+IN and CK+IN treatment(P≤0.05),COI1 was down-regulated from 24 to 144 h post-inoculated(P≤0.05),MYC2 was up-regulated at all time points except at 16 and 48 h post-inoculated(P≤0.05)at which there had no significant difference with CK+IN treatment.In JA+IN treatment,the expression of ICS was up-regulated at all time points except at 16 h post-inoculated(P≤0.05)at which the expression was down-regulated.The expression of PR1 had no significant difference from that in CK+IN treatment before 48 h,and then up-regulated(P≤0.05),while the expressions of AOS and MYC2 were up-regulated especially before 48 h post-inoculated(P≤0.05),and JAR1 had no significant difference from that in CK+IN treatment except up-regulated at 24 h.In addition,the expression variation trends of NPR1 and COI1 were consistent with those in SA+IN treatment.The results showed that D.rosae inhibited the synthesis of salicylic acid and the active jasmonic acid.SA+IN treatment was able to increase the expressions of genes in salicylic acid signaling pathway and promote the synthesis of jasmonic acid,but had little effect on the synthesis of active jasmonic acid.Meanwhile SA+IN treatment weakened the induction effect of D.rosae on MYC2 before 48 h post-inoculation,while the treatment increased the expressions of both ICS and PR1 as well as the expression of MYC2 after 48 h post-inoculated.JA+IN treatment not only promoted synthesis of active jasmonic acid and the expression PR1 but also increased the expression of MYC2.In addition,this study also found that in the SA+NO and JA+NO treatments,the expression levels of 7 genes changed to different degrees compared with that in CK treatments,but the change trends were inconsistent with the corresponding SA+IN and JA+IN treatments,indicating that D.rosae affected the induced expression of genes of the host by exogenous salicylic acid and jasmonic acid on the corresponding signaling pathway.【Conclusion】In conclusions,D.rosae has a strong inhibitory effect on the salicylic acid and jasmonic acid/ethylene signaling pathways of rose,and mainly inhibits salicylic acid signaling pathway before 48 h post-inoculation,and mainly inhibits the jasmonic acid/ethylene signaling pathway after 48 h post-inoculated.The pathogen,D.rosae,may realize dual inhibition of salicylic acid and jasmonic acid/ethylene signaling pathways of rose by enhancing the expression of MYC2 in roses.