环烷基磺酰胺类化合物抑制植物病原真菌作用机制研究

Sulfonylureas fungicides, as the highly active agents developed recently abroad to Oomycetes were received extensive attention because of its novel structure and lesser commercial agents. Scores of sulfonylureas compounds synthesized currently possess broad-spectrum antibacterial activity but Sulfonylureas compound SYAUP108 has systemic and broad-spectrum activity, especially against Botrytis cinerea in vivo and vitro. Their mechanism against plant pathogenic fungi is still unknown. The discovery of the mechanism of bioactive compounds in the early stages of research greatly accelerates the procedure for the development of new crop protection agents. Modern selective fungicide resistance is due to the drug target gene mutation. By this project, we intend to induce resistant mutants by repeated exposure to SYAUP108 or ultraviolet radiation treatment. The difference of whole-genome sequences between SYAUP108-sensitivive and SYAUP108-resistant isolates will be compared and analysised to identify the possible resistance target genes and mutations. Every mutations gene of SYAUP108-resistant isolate was knocked out and transferred into SYAUP 108-resistant isolate at their site to the target gene. The target gene will be cloned and expressed in soluble form in E. coli. The expressed proteins will be purified. The in vitro binding characteristics of these proteins with SYAUP108 in sensitive and resistant isolates will be studied and compared by fluorescence quenching to illustrate the mechanism of SYAUP 108 against plant pathogenic fungi. This study will provide some theory basis and driving force for the research and development of sulfonylureas compounds.

磺酰胺类杀菌剂是近年来国外开发的高效杀卵菌药剂，因其结构新颖，商品化产品较少，而日益受到人们关注。国内外研究者已合成数十种磺酰胺类化合物，杀菌谱异于商品化品种。本实验室合成的系列磺酰胺类化合物杀菌谱广，其中SYAUP108化合物具有内吸性，对灰葡萄孢表现出较高的离体和活体抑制活性。目前，磺酰胺类活性化合物作用机理尚未见报到，而活性化合物作用机理的早期研究将促进该类化合物的筛选和研发。现代选择性杀菌剂抗性大多由药剂作用靶标位点突变而产生。本研究拟采用灰葡萄孢抗药性菌株，比较药剂作用下敏感与抗性菌株形态初步确定药剂作用部位。比较抗性与敏感菌株全基因组序列，寻找差异基因和突变位点；对差异基因敲除和回复，明确相关的靶标基因；对靶标基因进行原核表达和蛋白纯化，研究靶标蛋白与药剂体外结合能力，阐明药剂的作用机理和作用靶标。本项研究必将为该类化合物进一步研发、创制提供理论依据和动力。

磺酰胺类杀菌剂是近年来开发的高效杀卵菌药剂，结构新颖，商品化产品较少，受到人们广泛关注。国内外研究者已合成数十种磺酰胺类化合物，抗菌谱异于商品化药剂，推测其可能具有独特的作用机理。本实验室合成的系列磺酰胺类化合物杀菌谱广，其中1S,2R-((3-溴苯乙基）氨基)-N-(4-氯-2-三氟甲基苯基)环己烷基-1-磺酰胺(SYAUP-CN-26)对灰葡萄孢表现出更优的离体和活体活性。为探明作用机理，本项目借助抗药性菌株从以下几个方面进行研究：（1）灰葡萄孢对化合物SYAUP-CN-26 生物学性状研究及抗药性菌株诱导；（2）药剂对抗药性菌株和敏感性菌株菌体形态和超微结构的影响比较；（3）灰葡萄孢抗药性相关功能基因筛选。（4）从蛋白水平对该药剂作用机制进行研究。结果表明：（1）SYAUP-CN-26可抑制多种植物病原真菌的菌丝生长和孢子萌发，尤以对灰葡萄孢Botrytis cinerea 的抑制活性较强，EC50 值分别为1.82 μg/mL 和14.98 μg/mL。20μg/mL的SYAUP-CN-26 能显著降低灰葡萄孢的产孢量、产菌核量，增加单菌核重，降低孢子及菌丝致病力。5株抗药性可稳定遗传的菌株适合度低于敏感菌株。（2）SYAUP-CN-26使灰葡萄孢菌敏感菌菌丝分枝增多，菌丝及孢子萎缩、塌陷、变形；菌体细胞壁外周物质增多，细胞膜结构受破坏，细胞空泡化严重，线粒体结构和嵴形态改变，药剂对抗药性菌影响较小或无影响；不影响细胞壁形成，但破坏细胞膜组成和功能，抑制菌体呼吸代谢和ATP合成，其中显著抑制复合体Ⅰ、复合体Ⅱ、复合体Ⅳ和复合体Ⅴ的活性，但对复合体Ⅲ无影响。（3）SYAUP-CN-26与腐霉利具有正交互抗药性，抗药性菌株组氨酸激酶活性高于敏感菌株，4株中抗菌株BcOS1基因的氨基酸序列均发生突变，即第348位氨基酸脯氨酸突变为亮氨酸（P348L），初步确定为抗药性功能基因。（4）蛋白质组分析结果表明药剂处理后差异蛋白亚细胞主要定位在胞浆蛋白、细胞膜和线粒体中。.本项目研究结果表明磺酰胺类化合物SYAUP-CN-26主要影响菌体质膜结构和功能，作用靶点推测与BosI基因有关。该项研究为该类化合物的进一步开发提供了重要的理论依据。