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稻瘟菌二肽基肽酶V基因DppV的功能分析
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引用本文:聂燕芳,冷梅钦,李冠军,李洁玲,李华平,李云锋.稻瘟菌二肽基肽酶V基因DppV的功能分析.植物保护学报,2021,48(4):712-722
DOI:10.13802/j.cnki.zwbhxb.2021.2020204
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作者单位E-mail
聂燕芳 华南农业大学制药工程系, 广州 510642
华南农业大学广东省微生物信号与作物病害重点实验室, 广州 510642 
 
冷梅钦 华南农业大学广东省微生物信号与作物病害重点实验室, 广州 510642
华南农业大学植物病理学系, 广州 510642 
 
李冠军 华南农业大学广东省微生物信号与作物病害重点实验室, 广州 510642
华南农业大学植物病理学系, 广州 510642 
 
李洁玲 华南农业大学广东省微生物信号与作物病害重点实验室, 广州 510642
华南农业大学植物病理学系, 广州 510642 
 
李华平 华南农业大学广东省微生物信号与作物病害重点实验室, 广州 510642
华南农业大学植物病理学系, 广州 510642 
 
李云锋 华南农业大学广东省微生物信号与作物病害重点实验室, 广州 510642
华南农业大学植物病理学系, 广州 510642 
yunfengli@scau.edu.cn 
中文摘要:为明确二肽基肽酶V(dipeptidyl-peptidase V,DppV)在稻瘟菌Magnaporthe oryzae中的生物学功能,采用在线软件对稻瘟菌DppV进行生物信息学分析,利用实时荧光定量PCR(quantitativereal-time PCR,qRT-PCR)技术分析其在稻瘟菌分生孢子不同萌发时间的表达量,通过基因敲除和功能回补、致病力测定等方法对其功能进行分析。结果表明,稻瘟菌DppV蛋白含有信号肽,定位于胞外,不含跨膜结构域,没有GPI锚定位点,为经典分泌蛋白,含DAP2结构域;其与小麦顶囊壳Gaeumannomyces tritici、早熟禾巨座壳Magnaporthiopsis poae、新烟曲霉Aspergillus novofumigatus和巴斯德伊蒙菌Emergomyces pasteurianus蛋白序列的相似度较高,分别为68.92%、68.41%、51.09%和49.96%;在稻瘟菌分生孢子萌发早期DppV基因均有表达,但当萌发时间为24 h时,其相对表达量最高,为5.61;稻瘟菌DppV基因敲除突变体菌株的菌落生长速度明显下降,产孢量(8.18×104个/cm2)和4个产孢相关基因MoHox2、MoCon8、MoSec22MoCos1的相对表达量显著降低,分生孢子萌发率(56.21%)明显下降,对SDS、NaCl、山梨醇和H2O2胁迫更加敏感,对水稻的致病力显著降低,水稻植株内真菌生物量明显减少,而稻瘟菌DppV基因回补菌株的表型、耐胁迫能力和致病力等则恢复到稻瘟菌野生型菌株的水平。表明DppV基因在稻瘟菌营养生长、产孢、致病力、对外界胁迫的应答及其在水稻体内的定殖等方面起着重要作用。
中文关键词:稻瘟菌  二肽基肽酶V  基因敲除  产孢量  致病力  胁迫  萌发率
 
Functional analysis of dipeptidyl-peptidase V gene DppV in rice blast fungus Magnaporthe oryzae
Author NameAffiliationE-mail
Nie Yanfang Department of Pharmaceutical Engineering, South China Agricultural University, Guangzhou 510642, Guangdong Province, China
Guangdong Province Key Laboratory of Microbial Signals and Disease Control, South China Agricultural University, Guangzhou 510642, Guangdong Province, China 
 
Leng Meiqin Guangdong Province Key Laboratory of Microbial Signals and Disease Control, South China Agricultural University, Guangzhou 510642, Guangdong Province, China
Department of Plant Pathology, South China Agricultural University, Guangzhou 510642, Guangdong Province, China 
 
Li Guanjun Guangdong Province Key Laboratory of Microbial Signals and Disease Control, South China Agricultural University, Guangzhou 510642, Guangdong Province, China
Department of Plant Pathology, South China Agricultural University, Guangzhou 510642, Guangdong Province, China 
 
Li Jieling Guangdong Province Key Laboratory of Microbial Signals and Disease Control, South China Agricultural University, Guangzhou 510642, Guangdong Province, China
Department of Plant Pathology, South China Agricultural University, Guangzhou 510642, Guangdong Province, China 
 
Li Huaping Guangdong Province Key Laboratory of Microbial Signals and Disease Control, South China Agricultural University, Guangzhou 510642, Guangdong Province, China
Department of Plant Pathology, South China Agricultural University, Guangzhou 510642, Guangdong Province, China 
 
Li Yunfeng Guangdong Province Key Laboratory of Microbial Signals and Disease Control, South China Agricultural University, Guangzhou 510642, Guangdong Province, China
Department of Plant Pathology, South China Agricultural University, Guangzhou 510642, Guangdong Province, China 
yunfengli@scau.edu.cn 
Abstract:In order to explore the biological function of dipeptidyl-peptidase V (DppV) in rice blast fungus Magnaporthe oryzae, bioinformatics analysis of its amino acid sequence characteristics was performed using on-line software. Quantitative real-time PCR (qRT-PCR) was used to quantify the mRNA expression levels of DppV in M. oryzae at different stages of conidia germination of M. oryzae. The function of DppV in M. oryzae was investigated by using gene deletion, complementation experiments and pathogenicity test. The results showed that DppV in M. oryzae was a typical secreted protein with an N-terminal signal peptide, extracellular localization, no transmembrane domain and no GPI-anchor site, and contained a highly conserved DAP2 domain. The amino acid sequence of DppV in M. oryzae was highly similar to those of Gaeumannomyces tritici (68.92% similarity), Magnaporthiopsis poae (68.41% similarity), Aspergillus novofumigatus (51.09% similarity) and Emergomyces pasteurianus (49.96% similarity). DppV in M. oryzae was transcribed at all stages of conidia germination of M. oryzae, and reached the highest level (5.61) at 24 h. DppV in M. oryzae deletion mutants (ΔMoDppV) showed obvious phenotypic defects, including reduction in colony growth, sporulation quantity (8.18×104 spores/cm2) and conidial germination (56.21%) compared to the wild-type strain ZC13. Relative expression levels of four conidiation-related genes (MoHox2, MoCon8, MoSec22 and MoCos1) were significantly decreased in ΔMoDppV strain. Deletion of DppV in M. oryzae also increased the sensitivity of M. oryzae to external stresses (SDS, NaCl, sorbitol and H2O2). Furthermore, ΔMoDppV showed a significant reduction in virulence and fungal biomass in rice seedlings. The complementation strain ΔMoDppV-com was able to restore all the defects of ΔMoDppV to a comparable level to the wild-type strain. Taken together, these results indicated that DppV played an important role in the mycelium growth, conidiation, tolerance to different stresses, colonization of rice plants and pathogenicity in M. oryzae.
keywords:Magnaporthe oryzae  dipeptidyl-peptidase V  gene deletion  conidiation  pathogenicity  stress  germination rate
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