萜类物质影响指状青霉麦角固醇合成的分子机制解析

Green mold caused by Penicillium digitatum is one of the most damaging fungal diseases in citrus fruit. Ergosterols serve an indispensible role in maintaining cell function and integrity in fungal. Previous studies and our former studies both revealed that, terpenoids can remarkably decrease the ergosterol content of P. digitatum and other pathogens, while the underlying mechanism remains undetermined. In the present research, the effects of different types of terpenoids (citronellal, α-terpineol and eugenol) on membrane permeability and ergosterol content of P. digitatum will be determined. The sterols in the mycelia of P. digitatum after the addition of terpenoids will be identified and analyzed by gas chromatography-mass spectrometry (GC-MS). Meanwhile, the quantitative real-time PCR will be conducted to determine the expression levels of essential genes involved in ergosterol biosynthetic pathway and therefore uncover the potential target site. The exogenous ergosterol will also be added to the culture media, in an effort to clarify its effects on the inhibition efficiency and the gene expression levels involved in ergosterol biosynthesis. To obtain the membrane proteins that may regulate ergosterol biosynthesis, the isobaric tags for relative and absolute quantization (iTRAQ) technique will be conducted to uncover the differentially expressed membrane proteins after the addition of terpenoids. All these are to explore the potential mechanism that terpenoids affect the ergosterol biosynthesis of P. digitatum. Our present research can not only provide a good opportunity for better understanding of antifungal mechanism of terpenoids, but also has important theoretical guidance for the development of novel terpenoids fungicides in the future.

指状青霉侵染果实引起的绿霉病是最严重的柑橘采后真菌病害。麦角固醇对维持真菌细胞膜功能和结构具有重要作用。前人和前期研究发现，萜类物质能显著降低指状青霉等致病菌麦角固醇含量，但对应的作用机制尚不清楚。本研究拟以指状青霉抑霉唑抗性和敏感菌株为对象，分析不同类型萜类物质（香茅醛、α-松油醇和丁香酚）对菌丝体细胞膜通透性和麦角固醇的影响；通过GC-MS鉴定和分析萜类物质处理对菌丝体固醇中间代谢物种类和含量的影响；通过荧光定量PCR检测麦角固醇合成必需基因的表达，揭示潜在的作用位点；分析外源麦角固醇添加对萜类物质抑菌性能和麦角固醇合成基因表达的影响，通过iTRAQ技术分析和鉴定受萜类影响的差异表达膜蛋白，挖掘可能参与调控麦角固醇代谢的蛋白类型；旨在揭示萜类物质影响指状青霉麦角固醇合成的作用机理。项目的开展不仅能丰富人们对萜类物质抑菌机理的研究，也为今后萜类抑菌剂的开发提供重要的理论指导。

指状青霉侵染引果实引起的绿霉病是最严重的柑橘真菌病害。麦角固醇对维持真菌细胞膜功能和结构具有重要作用。本研究探讨了植物精油组分香茅醛、α-松油醇和丁香酚对指状青霉抑霉唑敏感菌株（Pds01）和抗性菌株（Pdw03）麦角固醇合成的影响及潜在分子机制。抑菌结果显示：香茅醛、丁香酚和α-松油醇均能有效抑制Pds01和Pdw03菌丝体生长，但香茅醛对Pds01的MIC和MFC（分别为1.60 μL/mL和3.20 μL/mL）显著低于Pdw03（MIC和MFC均为6.40 μL/mL）；外源添加麦角固醇能不同程度减缓香茅醛、丁香酚和α-松油醇对Pds01和Pdw03菌株的抑制和细胞膜通透性伤害。GC-MS结果显示，香茅醛、丁香酚和α-松油醇均不同程度影响Pds01和Pdw03菌株麦角固醇生物合成途径中间代谢产物种类和含量，但不同组分调控位点存在差异；外源添加麦角固醇可不同程度恢复Pds01和Pdw03菌株麦角固醇含量。荧光定量PCR结果显示：香茅醛、丁香酚和α-松油醇处理后，指状青霉Pds01和Pdw03菌株13个麦角固醇合成基因（ERG1、ERG2、ERG3、ERG4、ERG5、ERG6、ERG7、ERG9、ERG11、ERG24、ERG25、ERG26和ERG27）表达出现明显变化，仅香茅醛组上述基因出现明显的抑制; 外源麦角固醇添加能恢复上述基因表达。膜蛋白组分析结果显示，香茅醛处理、外源添加麦角固醇+香茅醛处理后Pds01菌株差异膜蛋白共计372个，主要涉及到β-丙氨酸代谢、类固醇生物合成、萜类骨架生物合成等途径；麦角固醇代谢途径ERG2、ERG3、ERG4、ERG5、ERG6、ERG11、ERG24、ERG25与ERG26均为膜蛋白，其中ERG3、ERG4 、ERG25与ERG26表达水平发生明显改变。差异膜蛋白定量PCR结果显示，ERG3可能为香茅醛抑制指状青霉菌丝体麦角固醇合成途径的靶标位点，转录因子SreA基因可能参与麦角固醇补偿机制。在此基础上构建了指状青霉△SreA、△ERG3A和△ERG3B基因突变体。综上，本文研究结果表明，香茅醛能通过影响指状青霉麦角固醇合成来抑制指状青霉生长，作用靶标为ERG3，丁香酚和α-松油醇则通过其他作用方式抑制指状青霉生长，这一结果可为研究植物精油抑菌机理提供新的理论参考。