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小菜蛾Toll-6基因的克隆及功能分析
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引用本文:张姗姗,贾元虹,李金洋,林俊涵,尤民生,夏晓峰.小菜蛾Toll-6基因的克隆及功能分析.植物保护学报,2022,49(2):574-586
DOI:10.13802/j.cnki.zwbhxb.2021.2020194
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作者单位E-mail
张姗姗 福建农林大学应用生态研究所, 闽台作物有害生物生态防控国家重点实验室, 福州 350002
福建农林大学, 农业农村部闽台作物有害生物综合治理重点实验室, 教育部害虫生态防控国际合作联合实验室, 福州 350002 
 
贾元虹 福建农林大学应用生态研究所, 闽台作物有害生物生态防控国家重点实验室, 福州 350002
福建农林大学, 农业农村部闽台作物有害生物综合治理重点实验室, 教育部害虫生态防控国际合作联合实验室, 福州 350002 
 
李金洋 福建农林大学应用生态研究所, 闽台作物有害生物生态防控国家重点实验室, 福州 350002
福建农林大学, 农业农村部闽台作物有害生物综合治理重点实验室, 教育部害虫生态防控国际合作联合实验室, 福州 350002 
 
林俊涵 福建农林大学应用生态研究所, 闽台作物有害生物生态防控国家重点实验室, 福州 350002
福建农林大学, 农业农村部闽台作物有害生物综合治理重点实验室, 教育部害虫生态防控国际合作联合实验室, 福州 350002
福建生物工程职业技术学院, 福州 350002 
 
尤民生 福建农林大学应用生态研究所, 闽台作物有害生物生态防控国家重点实验室, 福州 350002
福建农林大学, 农业农村部闽台作物有害生物综合治理重点实验室, 教育部害虫生态防控国际合作联合实验室, 福州 350002 
msyou@fafu.edu.cn 
夏晓峰 福建农林大学应用生态研究所, 闽台作物有害生物生态防控国家重点实验室, 福州 350002
福建农林大学, 农业农村部闽台作物有害生物综合治理重点实验室, 教育部害虫生态防控国际合作联合实验室, 福州 350002 
xiaofengxia@fafu.edu.cn 
中文摘要:为探究Toll-6基因在小菜蛾Plutella xylostella先天免疫中的功能,对克隆鉴定的Toll-6基因序列进行生物信息学分析,利用实时荧光定量PCR检测Toll-6基因的时空表达模式及微生物侵染响应模式,体外验证其在微生物结合、病原相关分子模式(pathogen associated molecular pattern,PAMP)结合和细菌凝集等方面的功能。结果表明:Toll-6基因全长2 313 bp,编码770个氨基酸,预测Toll-6蛋白具有富含亮氨酸重复序列(leucine-rich repeats,LRR)胞外区、跨膜区和Toll/白介素-1受体同源(Toll/interleukin-1 receptor homologous,TIR)胞内等典型Toll受体家族特征;Toll-6基因在不同发育阶段及不同组织中均有表达,其中成虫期表达量最高,4龄幼虫期次之,在4龄幼虫中肠表达量最高;此外,苏云金芽胞杆菌Bacillus thuringiensis菌株Bt8010侵染小菜蛾4龄幼虫6 h后,Toll-6基因表达被显著抑制,而黏质沙雷氏菌Serratia marcescens菌株SmPXG6侵染小菜蛾4龄幼虫12 h时,Toll-6基因表达被显著上调,18 h后Toll-6基因表达被显著抑制;蛋白体外功能验证表明Toll-6蛋白不仅与细菌和真菌有直接结合能力,还能与肽聚糖和脂多糖结合,但不具备细菌凝集功能。表明Toll-6蛋白可能作为模式识别受体直接识别并结合入侵病原微生物表面的PAMP,启动小菜蛾对入侵病原微生物的先天免疫防御,且对不同微生物可能具有不同的响应机制。
中文关键词:小菜蛾  Toll受体  先天免疫  模式识别受体  基因表达  病原菌侵染
 
Cloning and functional characterization of Toll-6 gene in the diamond back moth Plutella xylostella
Author NameAffiliationE-mail
ZHANG Shan-shan State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Institute of Applied Ecology, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian Province, China
Joint International Research Laboratory of Ecological Pest Control, Ministry of Education 
 
JIA Yuan-hong State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Institute of Applied Ecology, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian Province, China
Joint International Research Laboratory of Ecological Pest Control, Ministry of Education 
 
LI Jin-yang State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Institute of Applied Ecology, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian Province, China
Joint International Research Laboratory of Ecological Pest Control, Ministry of Education 
 
LIN Jun-han State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Institute of Applied Ecology, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian Province, China
Joint International Research Laboratory of Ecological Pest Control, Ministry of Education
Fujian Vocational College of Bioengineering, Fuzhou 350002, Fujian Province, China 
 
YOU Min-sheng State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Institute of Applied Ecology, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian Province, China
Joint International Research Laboratory of Ecological Pest Control, Ministry of Education 
msyou@fafu.edu.cn 
XIA Xiao-feng State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Institute of Applied Ecology, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian Province, China
Joint International Research Laboratory of Ecological Pest Control, Ministry of Education 
xiaofengxia@fafu.edu.cn 
Abstract:In order to dissect the function of Toll-6 gene in the innate immunity of the diamond back moth Plutella xylostella, bioinformatics analysis of the cloned Toll-6 gene was performed, and the spatio-temporal expression pattern and microbial infection response pattern of Toll-6 gene was detected by quantitative real-time PCR; meanwhile, its functions in microbial binding, pathogen-associated molecular pattern(PAMP) binding and bacterial agglutination were verified in vitro. The results showed that the length of Toll-6 gene was 2 313 bp, encoding 770 amino acid residues, and the amino acid sequence contained leucine-rich repeats(LRR) extracellular region rich in leucine repeats, transmembrane region and Toll/interleukin-1 receptor homologous(TIR) intracellular region characterized by typical Toll receptor family. Toll-6 gene was expressed during different developmental stages, with the highest expression in adults, followed by the 4th-instar larvae, and in different tissues, with the highest expression in the midgut of the 4th-instar larvae. In addition, when the 4th-instar larvae of P. xylostella were infected with Bacillus thuringiensis strain Bt8010 for six hours, Toll-6 gene expression was significantly inhibited. In contrast, when the 4th-instar larvae of P. xylostella were infected with Serratia marcescens strain SmPXG6 for 12 h, Toll-6 gene expression was significantly up-regulated, but suppressed at 18 h after infection. Functional characterization of the protein in vitro demonstrated that Toll-6 protein could bind to bacteria and fungi, as well as peptidoglycan and lipopolysaccharide, although it did not function in bacterial agglutination. In conclusion, Toll-6 as a pattern recognition receptor(PRR) might directly recognize and bind to the PAMP, and initiate the innate immune defense against invasive pathogens in P. xylostella, although the response mechanisms varied with different microbes.
keywords:Plutella xylostella  Toll receptor  innate immunity  pattern recognition receptor  gene expression  pathogen infection
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