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可可毛色二孢β-1,4-葡聚糖内切酶LtEg1信号肽的鉴定及其酶活性分析
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引用本文:彭军波,李兴红,张玮,刘梅,邢启凯,燕继晔.可可毛色二孢β-1,4-葡聚糖内切酶LtEg1信号肽的鉴定及其酶活性分析.植物保护学报,2020,47(1):119-126
DOI:10.13802/j.cnki.zwbhxb.2020.2019045
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彭军波 北京市农林科学院植物保护环境保护研究所, 北方果树病虫害绿色防控北京市重点实验室, 北京 100097  
李兴红 北京市农林科学院植物保护环境保护研究所, 北方果树病虫害绿色防控北京市重点实验室, 北京 100097  
张玮 北京市农林科学院植物保护环境保护研究所, 北方果树病虫害绿色防控北京市重点实验室, 北京 100097  
刘梅 北京市农林科学院植物保护环境保护研究所, 北方果树病虫害绿色防控北京市重点实验室, 北京 100097  
邢启凯 北京市农林科学院植物保护环境保护研究所, 北方果树病虫害绿色防控北京市重点实验室, 北京 100097  
燕继晔 北京市农林科学院植物保护环境保护研究所, 北方果树病虫害绿色防控北京市重点实验室, 北京 100097 jiyeyan@vip.163.com 
中文摘要:为明确可可毛色二孢β-1,4-葡聚糖内切酶LtEg1是否能够分泌至胞外及其活性,采用酵母互补试验分析了内切酶LtEg1的信号肽活性,利用3,5-二硝基水杨酸法检测了内切酶LtEg1活性,通过实时荧光定量PCR测定了LtEg1基因在不同侵染阶段的表达量。内切酶LtEg1的信号肽和酵母蔗糖酶的融合表达能够引起酵母蔗糖酶的外泌,使得酵母转化子能够在以棉子糖为唯一碳源的培养基上生长。内切酶LtEg1能够以羧甲基纤维素钠为底物,发挥其酶活功能。与营养菌丝阶段相比,LtEg1基因在病原菌侵染阶段的表达水平显著升高;且在接种后48 h,LtEg1基因的表达量达到高峰,约为营养菌丝阶段的12倍,表明内切酶LtEg1作为重要的外泌蛋白酶,在可可毛色二孢侵染致病过程发挥了重要作用。
中文关键词:可可毛色二孢  β-1,4-葡聚糖内切酶  信号肽  酶活性
 
Identification of the signal peptide and enzymatic activity analysis of the endo-β-1,4-glucanase LtEg1 of fungal pathogen Lasiodiplodia theobromae
Author NameAffiliationE-mail
PENG Junbo Beijing Key Laboratory of Environment-Friendly Management of Diseases and Pests of North China Fruits, Institute of Plant and Environmental Protection, Beijing Academy of Agricultural and Forestry Sciences, Beijing 100097, China  
LI Xinghong Beijing Key Laboratory of Environment-Friendly Management of Diseases and Pests of North China Fruits, Institute of Plant and Environmental Protection, Beijing Academy of Agricultural and Forestry Sciences, Beijing 100097, China  
ZHANG Wei Beijing Key Laboratory of Environment-Friendly Management of Diseases and Pests of North China Fruits, Institute of Plant and Environmental Protection, Beijing Academy of Agricultural and Forestry Sciences, Beijing 100097, China  
LIU Mei Beijing Key Laboratory of Environment-Friendly Management of Diseases and Pests of North China Fruits, Institute of Plant and Environmental Protection, Beijing Academy of Agricultural and Forestry Sciences, Beijing 100097, China  
XING Qikai Beijing Key Laboratory of Environment-Friendly Management of Diseases and Pests of North China Fruits, Institute of Plant and Environmental Protection, Beijing Academy of Agricultural and Forestry Sciences, Beijing 100097, China  
YAN Jiye Beijing Key Laboratory of Environment-Friendly Management of Diseases and Pests of North China Fruits, Institute of Plant and Environmental Protection, Beijing Academy of Agricultural and Forestry Sciences, Beijing 100097, China jiyeyan@vip.163.com 
Abstract:To investigate the exocrine characteristics and enzymatic activity of the endo-β-1,4-glucanase LtEg1 of fungal pathogen Lasiodiplodia theobromae, the signal peptide of LtEg1 was identified by yeast complementary assay, and the enzymatic activity of LtEg1 was examined by using the 3,5-dinitrosalicylic acid method. Furthermore, the transcriptional profiles of LtEg1 gene at different infectious stages were detected by real-time fluorescent quantitative PCR. Yeast complementary investigation showed that fusion expression of the signal peptide of LtEg1 with yeast invertase that lacked its own signal peptide induced the secretion of invertase and afterwards, enabled yeast transformants to grow on media with raffinose as the sole carbon resource. Moreover, enzymatic activity tests indicated that LtEg1 protein could degrade the sodium carboxymethyl cellulose. The results of real-time fluorescent quantitative PCR showed that transcription levels of LtEg1 gene at infectious stages were significantly higher than that in vegetative hyphae. Besides, the transcription level of LtEg1 gene almost reached the peak at 48 h post inoculation, which was nearly 12 times as high as that of vegetative hyphae, indicating that LtEg1, as an important exocrine protease, played significantly roles during the infection process of L. theobromae.
keywords:Lasiodiplodia theobromae  endo-β-1,4-glucanase  signal peptide  enzymatic activity
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