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柑橘黄龙病菌过氧化物还原酶基因CLasPrx的克隆及功能分析
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引用本文:左溪如,王淘,陈烨,闫亚娜,黄桂艳,李瑞民.柑橘黄龙病菌过氧化物还原酶基因CLasPrx的克隆及功能分析.植物保护学报,2024,51(3):654-662
DOI:10.13802/j.cnki.zwbhxb.2024.2023089
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作者单位E-mail
左溪如 赣南师范大学生命科学学院, 江西 赣州 341000  
王淘 赣南师范大学生命科学学院, 江西 赣州 341000  
陈烨 赣南师范大学生命科学学院, 江西 赣州 341000  
闫亚娜 赣南师范大学生命科学学院, 江西 赣州 341000  
黄桂艳 赣南师范大学生命科学学院, 江西 赣州 341000 huangguiyan@gnnu.edu.cn 
李瑞民 赣南师范大学生命科学学院, 江西 赣州 341000 liruimin@gnnu.edu.cn 
中文摘要:为解析柑橘黄龙病菌亚洲种Candidatus Liberibacter asiaticus(CLas)逃逸活性氧伤害的机理,通过克隆其过氧化物还原酶(peroxiredoxin,Prx)编码基因的全长序列,对CLasPrx蛋白序列进行生物信息学分析、多重比对及系统发育分析,采用实时荧光定量PCR方法检测CLasPrx基因在柑橘不同组织中的表达模式,并在本氏烟Nicotiana benthamiana叶肉细胞瞬时表达CLasPrx分析其编码蛋白的亚细胞定位,利用碘化钾法测定 CLasPrx 蛋白清除 H2O2的活性。结果显示,克隆得到的CLasPrx基因序列全长为534 bp,编码177个氨基酸;CLasPrx蛋白包含1个Redoxin保守结构域;多重比对分析发现CLasPrx蛋白活性位点含有保守基序PGAFTPTC;系统发育分析表明CLasPrx蛋白与近缘物种的同源蛋白聚为一类;CLasPrx基因在感染黄龙病柑橘树秋梢中的表达水平显著高于在春梢中的表达水平;CLasPrx定位于本氏烟叶肉细胞的细胞质基质中;过表达CLasPrx蛋白能够显著降低本氏烟叶肉细胞内H2O2的含量。表明CLasPrx可能参与柑橘黄龙病菌清除H2O2的过程。
中文关键词:柑橘黄龙病菌  过氧化物还原酶  表达分析  亚细胞定位  序列分析
 
Cloning and functional analysis of a peroxiredoxin gene, CLasPrx, from huanglongbing pathogen Candidatus Liberibacter asiaticus
Author NameAffiliationE-mail
Zuo Xiru College of Life Sciences, Gannan Normal University, Ganzhou 341000, Jiangxi Province, China  
Wang Tao College of Life Sciences, Gannan Normal University, Ganzhou 341000, Jiangxi Province, China  
Chen Ye College of Life Sciences, Gannan Normal University, Ganzhou 341000, Jiangxi Province, China  
Yan Yana College of Life Sciences, Gannan Normal University, Ganzhou 341000, Jiangxi Province, China  
Huang Guiyan College of Life Sciences, Gannan Normal University, Ganzhou 341000, Jiangxi Province, China huangguiyan@gnnu.edu.cn 
Li Ruimin College of Life Sciences, Gannan Normal University, Ganzhou 341000, Jiangxi Province, China liruimin@gnnu.edu.cn 
Abstract:To gain insights into the process of the huanglongbing pathogen Candidatus Liberibacter asiaticus (CLas) avoiding oxidative damage, the full-length sequence of the peroxiredoxin (Prx) gene CLasPrx was cloned and the encoded protein of the CLasPrx gene was analyzed through sequence analysis, multiple sequence alignment, and phylogenetic analysis. Additionally, the expression of the CLasPrx gene in various tissues was observed by real-time quantitative PCR, and its subcellular localization was determined by transient expression in mesophyll cells of Nicotiana benthamiana; the activity of CLasPrx in eliminating H2O2 was measured using potassium iodide method. Results showed that the CLasPrx gene was 534 bp in length and could encode a protein of 177 amino acids. A Redoxin domain was observed to be conserved in the CLasPrx protein, and multiple sequence alignment analysis indicated that the active site of the CLasPrx protein carries the conserved motif PGAFTPTC. Phylogenetic analysis indicated that the CLasPrx was clustered with the orthologous proteins of closely related species. The expression level of CLasPrx in the autumn shoots was significantly higher than in the spring shoots. Subcellular localization analysis indicated that CLasPrx localized in the cytoplasm of mesophyll cells of N. benthamiana. The level of H2O2 was significantly decreased due to the overexpression of the CLasPrx in N. benthamiana. This study revealed that CLasPrx might play an important role in the H 2O2 scavenging process in CLas.
keywords:Candidatus Liberibacter asiaticus  peroxiredoxin  expression analysis  subcellular localization  sequence analysis
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