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中国农学通报

中国农学通报 ›› 2023, Vol. 39 ›› Issue (27): 103-109.doi: 10.11924/j.issn.1000-6850.casb2022-0814

• 生物科学 • 上一篇    下一篇

甘薯黑斑病菌Cel61A基因克隆与表达分析

罗勤川(), 孙厚俊, 唐伟, 谢逸萍, 杨冬静, 马居奎, 陈晶伟, 高方园, 张成玲()   

  1. 江苏徐淮地区徐州农业科学研究所/中国农业科学院甘薯研究所/农业农村部甘薯生物学与遗传育种重点实验室，江苏徐州 221131
  • 收稿日期:2022-09-26 修回日期:2022-12-15 出版日期:2023-09-25 发布日期:2023-09-22
  • 通讯作者: 张成玲，女，1983年出生，山东沂源人，副研究员，研究生，博士，主要从事甘薯真菌致病相关基因挖掘和功能分析。通信地址：221131 江苏省徐州市贾汪区鲲鹏路高铁站北徐州农科院科研楼327，Tel：0516-082189220，E-mail：zhchlig5291@163.com。
  • 作者简介:

    罗勤川，男，1994年出生，四川遂宁人，硕士研究生，主要从事甘薯病虫害研究。通信地址：221131 江苏省徐州市贾汪区鲲鹏路高铁站北徐州农科院科研楼327，Tel：0516-082189220，E-mail：。

  • 基金资助:
    国家甘薯产业技术体系“线虫与真菌病防控”(CARS-10-GW17); 国家重点研发计划资助“杂粮作物抗生物逆境遗传机制与调控网络”(2018YFD1000703); “杂粮作物抗逆和品质形成与调控”(2018YFD1000700); 江苏省六大高峰人才项目“甘薯黑斑病致病机理及综合防控技术”(NY-202)

Cel61A Gene in Ceratocystis fimbriata: Cloning and Expression Analysis

LUO Qinchuan(), SUN Houjun, TANG Wei, XIE Yiping, YANG Dongjing, MA Jukui, CHEN Jingwei, GAO Fangyuan, ZHANG Chengling()   

  1. Xuzhou Institute of Agricultural Sciences in Jiangsu Xuhuai Area/ Sweet Potato Research Institute of Chinese Academy of Agricultural Sciences/ Key laboratory of Biology and Genetic Improvement of Sweet potato, Ministry of Agriculture and Rural Affairs, Xuzhou, Jiangsu 221131
  • Received:2022-09-26 Revised:2022-12-15 Published-:2023-09-25 Online:2023-09-22

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摘要：

为明确甘薯长喙壳菌致病相关基因多糖单加氧酶基因(CfCel61A)在病菌侵染甘薯中的表达模式及编码蛋白的理化特性。以11株来自不同地区的菌株为材料，克隆并利用生物信息学软件明确CfCel61A蛋白理化性质，用qRT-PCR分析基因表达。结果表明，CfCel61A编码区均为1155 bp，一致率99.5%~100%，编码384个氨基酸，一致率为98.4%~100%；分子量为34.49 kDa，等电点为5.59，亲水系数为-0.195，是稳定的亲水蛋白；N端有一个信号肽，亚细胞定位在核内，无跨膜结构；含58个磷酸化和2个糖基化位点，蛋白二级结构无规则卷曲占比最高，为69.01%。CfCel61A在病菌侵染中表达量显著上调，但不同侵染阶段上调幅度不同，侵染‘南京92’和‘徐薯37’后分别在12 h和6 h达到最高。综上，CfCel61A在C. fimbriata侵染甘薯中可能发挥重要作用，可为病害药剂开发提供新靶标。

关键词: 甘薯长喙壳菌, 多糖单加氧酶, 生物信息学, 克隆, 基因表达

Abstract:

The aim was to clarify the expression pattern and the physicochemical characteristics of the encoded protein of polysaccharide monooxygenase gene (CfCel61A), a pathogenic gene of Ceratocystis fimbriata infecting sweet potato. Eleven strains from different regions were used as materials, CfCel61A was cloned, and the physicochemical properties of CfCel61A were determined by bioinformatics analysis software. The gene expression was analyzed by qRT-PCR. The results showed that the coding region of CfCel61A was 1155 bp with a concordance rate of 99.5%-100%, and it encoded 384 amino acids with the concordance rate of 98.4%-100%. The predicted molecular weight was 34.49 kDa, the isoelectric point was 5.59, the hydrophilicity coefficient was -0.195，and it was a stable hydrophilic protein. There was a signal peptide at the N-terminal, and the subcellular localization was in the nucleus without transmembrane structure. There were 58 phosphorylation sites and 2 potential glycosylation sites. The irregular curl of protein secondary structure accounted for 69.01%, which was the highest. The expression of CfCel61A was significantly up-regulated during the infection of C. fimbriata, while the up-regulation ranges were varied at different stages of infection, and it reached the highest level at 12 h and 6 h after the infection in 'Nanjing 92' and 'Xushu 37', respectively. In short, CfCel61A in C. fimbriata may play an important role in the infection of sweet potato and the result will provide a new target for drug development.

Key words: Ceratocystis fimbriata, polysaccharide monooxygenase, bioinformatics, clone, gene expression