西瓜噬酸菌铜代谢相关基因copZ功能分析  

Functional analysis of copZ involved in copper metabolism of Acidovorax citrulli

作　　者：刘堰凤 关巍 王铁霖[3] 李展鸿 杨玉文[2] 刘慧芹 赵廷昌[2] LIU Yanfeng;GUAN Wei;WANG Tielin;LI Zhanhong;YANG Yuwen;LIU Huiqin;ZHAO Tingchang(Tianjin Agricultural University,College of Horticulture and Landscape,Tianjin 300384,China;State Key Laboratory for Biology of Plant Diseases and Insect Pests,Institute of Plant Protection,Chinese Academy of Agricultural Sciences,Beijing 100193,China;State Key Laboratory Breeding Base of Dao-di Herbs,National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China)

机构地区：[1]天津农学院园艺园林学院,天津300384 [2]中国农业科学院植物保护研究所/植物病虫害生物学国家重点实验室,北京100193 [3]中国中医科学院中药资源中心/道地药材国家重点实验室培育基地,北京100700

出　　处：《植物病理学报》2022年第3期377-387,共11页Acta Phytopathologica Sinica

基　　金：天津市企业科技特派员项目(20YDTPJC01330);国家重点研发计划(2018YFD0201300);现代农业产业技术体系(CARS-26);中国农业科学院创新团队项目;中央本级重大增减支项目(2060302);天津市高校中青年骨干创新人才培养计划(J01009030709);天津市研究生科研创新项目(2019XY017)。

摘　　要：瓜类细菌性果斑病(Bacterial fruit blotch,BFB)病原菌为西瓜噬酸菌(Acidovorax citrulli,Ac),主要为害西瓜、甜瓜等葫芦科作物,是瓜类生产上主要细菌性病害。目前生产上对该病害的防治手段主要以化学防治为主,其中铜制剂的防治效果较为显著。本研究以西瓜噬酸菌野生型菌株Aac-5中铜代谢相关基因copZ为对象,探究copZ基因缺失对西瓜噬酸菌抗铜性、生长能力等表型的影响;采用RT-qPCR技术测定在不添加和添加外源铜离子条件下,基因copZ缺失后对其他铜代谢相关基因表达量的影响。成功构建了西瓜噬酸菌copZ基因缺失突变菌株ΔcopZ和互补菌株ΔcopZ-comp。结果表明,野生型菌株Aac-5与ΔcopZ-comp菌株在添加1.25 mmol·L^(-1)CuSO_(4)的生长能力与不添加CuSO_(4)相似,而ΔcopZ菌株的生长能力减弱;当添加CuSO_(4)至2.50 mmol·L^(-1)时,野生型菌株Aac-5、ΔcopZ菌株和ΔcopZ-comp菌株的生长能力均减弱。基因copZ缺失在不含铜和含铜条件下均能使西瓜噬酸菌生物膜形成能力减弱;在不含铜条件下基因copZ不影响西瓜噬酸菌的运动能力,在含有1.25 mmol·L^(-1)CuSO_(4)的运动性培养基中各菌株运动能力丧失。ΔcopZ菌株在西瓜子叶内的生长能力相较于野生型菌株Aac-5显著增强,但在西瓜幼苗上的致病力差异不显著,仍具有引起烟草过敏性反应的能力。在不同铜浓度处理后,铜代谢相关基因cueR、copA表达量在野生型菌株中的变化趋势与ΔcopZ菌株存在差异;基因cusA、hylD在野生型菌株中表达量变化趋势与ΔcopZ菌株一致。这表明基因copZ在西瓜噬酸菌铜代谢过程中具有重要作用。Bacterial fruit blotch(BFB)of watermelon and melon,caused by Acidovorax citrulli(Ac),damages watermelon,melon and other cucurbit crops being a threat to cucurbits industry.At present,the control methods of BFB are mainly based on chemicals,among which the copper-based bactericides have the most significant control effect.The copZ gene plays critical roles in the copper-metabolism of A.citrulli.In this study,to explore the functions of copZ in A.citrulli,we used A.citrulli Aac-5 as the wild-type strain to construct mutant strainΔcopZ,and its complementary strainΔcopZ-comp.The copper resistance,pathogenicity,mobility and hypersensitive response of the subsequent strains were determined using tobacco plants..In addition to the phenotypes,we also analyzed the expression levels of the selected genes in the mutant strain by RT-qPCR,including those related to copper-metabolism genes.The results showed that the copZ deletion mutant strainΔcopZ and complementary strainΔcopZ-comp of A.citrulli strain Aac-5 were successfully constructed.The growth ability of wild-type strain Aac-5 and complementary strainΔcopZ-comp incubated in KB liquid medium with 1.25 mmol·L^(-1)CuSO_(4)was similar to that of the two strains incubated without CuSO_(4),while the growth ability of mutant strainΔcopZ was significantly decreased when 1.25 mmol·L^(-1)CuSO_(4)was added into KB liquid medium.When incubated in KB liquid medium with 2.50 mmol·L^(-1)CuSO_(4),the growth ability of Aac-5,ΔcopZ andΔcopZ-comp was significantly decreased.The biofilm formation of A.citrulli strains was decreased when incubated in KB liquid medium with 0 mmol·L^(-1),1.25 mmol·L^(-1)and 2.50 mmol·L^(-1)CuSO_(4).The elimination of copZ did not affect the swimming ability of A.citrulli,which were lost when copper sulfate was added into medium.In our pathogenicity tests,the growth ability of mutant strainΔcopZ in watermelon cotyledons was significantly higher than that of wild-type strain Aac-5,but the difference in pathogenicity on watermelon seedlings was no

关 键 词：抗铜 西瓜噬酸菌 copZ 基因功能分析 

分 类 号：S651[农业科学—果树学]