肉桂醛介导H2O2双向调控链格孢菌非寄主选择性毒素合成机制

Alternaria alternata is one of major fungus causing the decay of mildew rot in grape fruits, it could generate mycotoxins during the fungi growth and result the harm to human health. Cinnamaldehyde, a kind of plant essential oils, which proved to be effectively against A. alternata, but further investigation on the regulations of mycotoxins biosynthesis was remain undetermined. This study will start with the effect of cinnamaldehyde on the inhibition of A. alternata mycotoxins biosynthesis, mainly focus on: 1) the optimization of HPLC for mycotoxins measurements, and will study the effect of cinnamaldehyde, cinnamaldehyde + H2O2, and cinnamaldehyde + DMTU treatment on the synthesis of Non-Host Specific Toxin (NHST) in A. alternata at different culturing time; 2) the transcriptome sequencing (RNA-seq) will be adapted to reveal the transcriptome profilings of A.alternata after different treatments; 3) the isobaric tags for relative and absolute quantitation (iTRAQ) techniques will be used for analysing the proteome profilings after A. alternata cultured with cinnamaldehyde, H2O2 and DMTU. 4) the quantitative real-time PCR will be conducted to confirm the correlation analysis results of transcriptome and proteome profilings, thereby to reveal the acting elements of transcription factor in the promoter of differential genes, and the molecular regulatory mechanism of H2O2 on the synthesis of Non-Host Specific Toxin. 5) will investigate the changeable enzymatic activity in the differential metabolic pathway (mainly focus on REDOX metabolic pathway), finally to reveal the bi-directional control mechanism of H2O2 on the synthesis of Non-Host Specific Toxin in Alternaria spp. treated Cinnamaldehyde. This study will interpret the regulation mechanism of NHST synthesis from physiology, biochemistry and molecular biology level after cinnamaldehyde, H2O2 and DMTU treatments, and the results of the study will provide a solid theoretical foundation on the effect of essential oils on mycotoxins synthesis.

链格孢菌是引起葡萄采后病害的主要病菌，其产生的真菌毒素对人体健康有巨大危害。前期研究发现肉桂醛可抑制链格孢菌生长，且H2O2可能参与调控肉桂醛对非寄主选择性毒素合成的影响。本项目主要研究：1）测定肉桂醛、肉桂醛+H2O2和肉桂醛+DMTU对非寄主选择性毒素合成的影响；2）利用RNA-seq和iTRAQ技术筛选不同处理间的差异基因、转录因子及受影响的代谢途径；3）利用荧光定量PCR验证基因表达并明确受影响的代谢途径，分析差异基因启动子序列，确定转录因子作用元件，从分子生物学的角度确定H2O2参与调控链格孢菌非寄主选择性毒素合成的方式；4）测定受影响代谢途径中的酶活性，从生理生化角度验证参与肉桂醛介导H2O2影响非寄主选择性毒素合成过程的代谢途径。研究结果从生理生化及分子生物学角度阐明肉桂醛介导H2O2对链格孢菌非寄主特异性毒素合成的双向调控机制，可为控制采后葡萄果实真菌毒素污染奠定理论基础。

本项目研究肉桂醛介导H2O2对链格孢菌非寄主特异性毒素合成的双向调控机制，主要研究：1）研究发现肉桂醛+H2O2（CIN+H2O2）处理促进链格孢毒素AOH和AME的合成；肉桂醛+DMTU（CIN+DMTU）可显著降低链格孢菌AOH和AME毒素的合成，而对TEA毒素呈现出极大的促进作用。初步推测低浓度肉桂醛（0.0125 μl/mL）可能通过影响菌体内H2O2水平对链格孢毒素的合成进行调控。2）利用RNA-seq筛选关键PKS酶基因和抗氧化酶（CAT、SOD、POD和GPx）基因并对其表达量进行分析。将8个与链格孢毒素合成途径相关的Pks基因（PksH、PksA、PksJ、PksF、PksC、PksE、PksD和PksG）进行qRT-PCR验证，结果显示CIN+H2O2处理84 h后可提高PksH、PksA、PksJ、PksF、PksC、PksE和PksG基因的表达量，CIN+DMTU处理84 h后可显著降低PksH、PksA、PksJ、PksF、PksC、PksE、PksD和PksG基因的表达量，验证的整体结果与转录组数据基本吻合，可初步推测肉桂醛介导H2O2调控链格孢毒素合成相关的基因为PksH、PksA、PksJ、PksF、PksC、PksE、PksD和PksG。3）CIN+H2O2处理后可致链格孢菌体内H2O2水平的升高，促使细胞产生氧化应激，导致ROS爆发和膜脂过氧化物MDA大量积累。过量的H2O2诱导上游CAT、SOD和GPx抗氧化酶基因的表达，清除过量ROS进而缓解菌体细胞的氧化损伤。同时，链格孢菌体内超氧阴离子自由基在SOD酶作用下转化为H2O2，造成致H2O2和MDA的大量积累，营造氧化胁迫环境从而有益于毒素的积累。然而，H2O2清除剂DMTU可降低菌体内ROS水平，抑制抗氧化酶基因的表达以及抗氧化酶的活性，缓解氧化胁迫作用不益于毒素的积累。本研究证明肉桂醛可通过影响链格孢菌体细胞内氧化还原状态，对抗氧化酶基因表达以及抗氧化酶活性进行调控，从而降低H2O2水平导致链格孢毒素合成受到抑制。