生防假单胞菌Pf-5对秀丽隐杆线虫的毒性及其作用机制  

Toxicity and nematicidal mechanism of Pseudomonas protegens Pf-5 against Caenorhabditis elegans

作　　者：娄志英 王云鹏[2] 袁启明 张露露 顾沁 王暄 伍辉军 高学文 LOU Zhiying;WANG Yunpeng;YUAN Qiming;ZHANG Lulu;GU Qin;WANG Xuan;WU Huijun;GAO Xuewen(College of Plant Protection/Key Laboratory of Integrated Management of Crop Diseases and Pests,Ministry of Education,Nanjing Agricultural University,Nanjing 210095,China;Jiangsu Provincial Key Construction Laboratory of Probiotics Preparation/Huaiyin Institute of Technology,Huai’an 223003,China)

机构地区：[1]南京农业大学植物保护学院/农作物生物灾害综合治理教育部重点实验室,江苏南京210095 [2]江苏省益生制剂重点建设实验室/淮阴工学院,江苏淮安223003

出　　处：《南京农业大学学报》2019年第5期827-834,共8页Journal of Nanjing Agricultural University

基　　金：国家重点研发计划项目(2017YFD0201101);江苏省自然科学基金项目(BK20181322);中央高校科研业务经费专项资金(KYZ201708)

摘　　要：[目的]本文旨在研究生防假单胞菌(Pseudomonas protegens)Pf-5菌株的杀线虫活性,以及鉴定其产生的主要杀线虫活性物质。[方法]采用基于sacB基因的蔗糖致死无标记突变体系,构建Pf-5菌株的相关突变体;以模式线虫秀丽隐杆线虫(Caenorhabditis elegans)为作用对象,利用绿色荧光蛋白基因对菌株进行标记,研究菌株对秀丽隐杆线虫的致死效果;利用扫描和透射电镜研究Pf-5菌株杀线虫物质的作用机制。[结果]Pf-5菌株对秀丽隐杆线虫具有很强的致死活性,用不同浓度的Pf-5菌液处理线虫,48h对秀丽隐杆线虫的致死中浓度(LC50)为4.92×10^7CFU·mL^-1,96hLC50为3.58×10^6CFU·mL^-1;处理48h时,野生型Pf-5菌株对秀丽隐杆线虫的致死率达到73.57%,而突变体Pf-5ΔfitD的致死活性显著下降,只有25.04%;Pf-5菌株fitD基因的Delivery结构域和CROPS结构域缺失突变体对线虫的致死活性也显著下降,表明FitD蛋白是Pf-5菌株的主要杀线虫物质。扫描电镜结果表明,Pf-5菌株及其突变体都不会破坏线虫的体壁;透射电镜的结果则表明,Pf-5菌株导致线虫肠道绒毛变薄和脱落,而突变体Pf-5ΔfitD则没有此效果。[结论]生防假单胞菌Pf-5菌株中杀线虫物质为FitD蛋白,初步表明其作用位点在肠道部位。[Objectives] This paper aimed to study the nematicidal activity of the Pseudomonas protegens Pf-5,and to identify the major nematicidal compounds produced by this strain.[Methods] The unmarked deletion mutants of Pf-5 were constructed based on sucrose-induced ( sacB gene)lethalality.The model nematodes Caenorhabditis elegans was used as the target.The strains were labeled with green fluorescent protein gene to study the lethal effect of the strain on this nematode.The possible action site of Pf-5 strain was observed by using scanning and transmission electron microscopy.[Results] The Pf-5 strain had strong lethal activity against C.elegans.The LC 50 of Pf-5 against C.elegans at 48 h and 96 h,was respectively 4.92×10^7 CFU·mL^-1 and 3.58×10^6 CFU·mL^-1.When treated C.elegans with Pf-5 for 48 h,the lethal rate was 73.57%,but when fitD gene was knocked out,the lethal activity of Pf-5Δ fitD was significantly reduced,only 25.04% compared to the wide type.When Delivery domain and CROPS domain of fitD gene were respectively deleted,the lethal activity of mutants also significantly decreased.This indicated that the FitD protein was the major nematicidal substance produced by Pf-5 strain.Scanning electron microscopy showed that Pf-5 strain and its mutant did not destroy the body wall of nematode.But when observed using transmission electron microscopy,the results had showed that the intestinal villi of nematode became thinner,and even fell off,but the mutant Pf-5Δ fitD didn’t have such effect.[Conclusions] The nematicidal substance in the P.protegens Pf-5 strain is the FitD protein,and its site of action is the intestinal tract.

关 键 词：生防假单胞菌Pf-5 fitD基因 杀虫毒素 秀丽隐杆线虫 

分 类 号：S435[农业科学—农业昆虫与害虫防治]