柠檬烯诱导指状青霉孢子萌发的作用机理

Green mold caused by Penicillium digitatum is one of the most damaging postharvest diseases in citrus . Both previous and our former studies revealed that, as the principal volatile component in citrus oil gland, limonene can remarkably induce the spore germination of P. digitatum, but the underlying mechanism remains undetermined. In the present research, the effects of limonene at different culture time on the spore germination of P. digitatum will be determined. The metabolism of the substances in spores of P. digitatum after the induction of limonene at different time will be identified and analyzed by gas chromatography−mass spectrometry (GC-MS). Meanwhile, the limonene contents in spore suspensions and in spores will be analyzed by gas chromatography (GC), in an effort to clarify the role of limonene. The transcriptome sequencing (RNA-seq) and isobaric tags for relative and absolute quantitation (iTRAQ) techniques will be adapted to reveal the transcriptomic and proteomic profiles when the spore germination of P. digitatum is induced by limonene. The quantitative real-time PCR will be further conducted to confirm the correlation analysis results of transcriptomic and proteomic profiles, thereby to reveal the metabolic pathway limonene involved in. All these are to explore the potential mechanism of limonene-induced spore germination of P. digitatum. Our present research can not only provide a good opportunity for better understanding of pathogenesis of green mold in citrus fruit, but also has important theoretical and practical guidance for controlling the green mold by changing the compositions and contents of volatile components in citrus fruit in the future.

指状青霉（Penicillium digitatum）侵染引起的绿霉病是最严重的柑橘采后病害。前人和前期研究均发现，柑橘油胞主要芳香组分柠檬烯能显著诱导指状青霉孢子萌发，但对应的作用机制尚不明了。本研究拟以指状青霉分生孢子为对象，分析不同时间柠檬烯诱导对孢子萌发的影响；通过气质联用（GC-MS）鉴定和分析不同时间柠檬烯诱导后孢子内物质代谢规律，通过气相色谱（GC）测定菌悬液和孢子内柠檬烯含量变化，明确柠檬烯的作用；通过转录组测序技术（RNA-seq）和同位素相对标记与定量技术（iTRAQ）获得指状青霉孢子对柠檬烯诱导做出应答的转录和蛋白组证据，关联后进行荧光定量PCR验证，揭示柠檬烯参与的代谢过程；旨在探讨柠檬烯诱导指状青霉孢子萌发的作用机理。项目的开展不仅能丰富人们对柑橘采后绿霉病害发病规律的认识，也为今后通过改变柑橘果实芳香物质含量和组成来控制采后绿霉病发生等具有重要的理论和现实意义。

由指状青霉侵染引起的绿霉病是最严重的柑橘采后病害之一。柑橘油胞主要芳香组分柠檬烯能显著诱导指状青霉分生孢子萌发，加速绿霉病害发生进程。本研究通过多组学技术探讨了柠檬烯诱导指状青霉孢子萌发的分子机制。结果表明，低浓度（0-0.50μL/mL）柠檬烯可以促进孢子萌发，高浓度（>1.0μL/mL）则抑制其萌发，最佳诱导浓度为0.25μL/mL；外源添加5μL 0.25-2.0μL/mL柠檬烯能加速绿霉病的发病过程。GC-MS分析结果显示，低浓度（0-0.50μL/mL）柠檬烯处理指状青霉孢子不会发生物质转换，但高浓度下(>1.0μL/mL)少量柠檬烯被转化为α-松油醇。GC结果显示，柠檬烯浓度达到0.50μL/mL后，孢内柠檬烯含量不再增加，0.25μL/mL柠檬烯诱导培养11h后孢内柠檬烯含量不再增加，且孢内外柠檬烯和与初始添加量无显著差异，说明柠檬烯很可能充当诱导指状青霉分生孢子萌发的信号物。GC-TOF-MS、RNA-seq和iTRAQ分析结果显示，0.25μL/mL柠檬烯诱导7h和9h后指状青霉TCA循环、ABC转运、代谢途径，次生代谢物合成、淀粉和糖代谢、MAKP信号转导和活性氧生成和代谢等多个过程受到显著影响。组学关联并经荧光定量PCR确认了19个差异表达基因和蛋白（12个基因表达上调，7个基因表达下调）。其中，SOD和CAT2基因的表达受到明显诱导，而CAT1和GST的基因表达受到抑制，说明ROS的生成和代谢参与了柠檬烯诱导的孢子萌发进程，CAT，SOD和GST酶活性结果以及ROS、H2O2和GSH含量变化进一步证实了此观点。根据研究结果，活性氧可能在诱导前期（5h前）就已经参与了指状青霉孢子萌发的调控。进一步通过RNA-seq分析了0.25μL/mL柠檬烯诱导3h和5h的指状青霉孢子转录组，发现谷胱甘肽代谢途径参与了柠檬烯诱导的孢子萌发进程；LC-QTOFMS分析结果显示，0.25μL/mL柠檬烯诱导3h后，多个与谷胱甘肽合成和代谢相关的代谢物含量发生显著变化；外源添加谷胱甘肽显著影响柠檬烯诱导的孢子萌发进程，内源谷胱甘肽含量与孢子萌发之间存在关联。综上，本文研究结果表明，柠檬烯诱导条件下谷胱甘肽可能通过调控指状青霉早期ROS稳态平衡来促进指状青霉孢子萌发，这一结果可为柑橘病害发生和防控提供新的理论基础。