| 辣椒炭疽病菌效应因子NIS1-PCR-RFLP标记系统的建立 |
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| 引用本文： | 欧阳超,刘思珍,满益龙,盛家伟,陈岳,张鑫,刘勇,张德咏,谭新球.辣椒炭疽病菌效应因子NIS1-PCR-RFLP标记系统的建立[J].南方农业学报,2021,52(9):2473-2481. |
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| 作者姓名： | 欧阳超 刘思珍 满益龙 盛家伟 陈岳 张鑫 刘勇 张德咏 谭新球 |
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| 作者单位： | 1 湖南大学研究生院隆平分院, 长沙 410125;2 湖南省农业科学院植物保护研究所, 长沙 410125;3 湖南省农业科学院生物技术研究所, 长沙 410125;4 湖南农业大学, 长沙 410000 |
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| 基金项目： | 国家自然科学基金面上项目（31972223）；国家自然科学青年基金项目（31701814）；湖南省农业科技创新资金项目（2020CX39，2020CX43） |
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| 摘 要： | 【目的】基于辣椒炭疽病菌种群复杂、田间复合侵染种群组成不清，导致防控相对困难的问题，建立一种快速直观的辣椒炭疽病菌区分标记系统，为其病害田间防控提供科学依据。【方法】利用rDNA-ITS通用引物克隆田间分离的22株辣椒炭疽病菌rDNA-ITS，通过其序列比对分析构建系统发育进化树，初步确定22株供试菌株的种类和分类地位；选用效应因子NIS1基因为靶标，根据辣椒胶孢炭疽菌（Colletotrichum gloeosporioides）和黄瓜炭疽菌（C.orbiculare）NIS1基因比对分析，设计NIS1基因简并引物，克隆22株辣椒炭疽病菌的NIS1基因，利用其序列比对分析构建系统发育进化树，分析比较rDNA-ITS和NIS1基因区分供试菌株的差异，进而对22株不同辣椒炭疽病菌NIS1基因进行分析，选择限制性内切酶，分析NIS1基因的PCR产物限制性片段多态性（RFLP）差异，建立NIS1-PCR-RFLP标记系统。【结果】rDNA-ITS序列系统进化分析表明22株辣椒炭疽病菌属于5种炭疽病菌，即胶孢炭疽菌（C.gloeosporioides）、短孢炭疽菌（C.brevisporum）、尖孢炭疽菌（C.acutatum）、平头炭疽菌（C.truncatum）和黑点炭疽菌（C.capsici），其序列同源性为85.6%~99.8%，但C.capsici和C.truncatum处于同一混合组群；22株辣椒炭疽病菌的NIS1基因两端序列较保守，中间存在可变区，与已报道的C.orbiculare的NIS1基因同源性在34.6%~78.9%；NIS1基因系统发育进化树能将22株辣椒炭疽病菌分成5个组群，C.capsici和C.truncatum处于不同组群，表明其区分度优于rDNA-ITS；NIS1-PCRRFLP主要差异片段显示C.gloeosporioides和C.brevisporum的最大片段均为210 bp，但片段数目不同，而C.acutatum为292 bp，C.capsici为685 bp，C.truncatum为345 bp，参照菌株C.orbiculare为497 bp，能够直观观察区分。【结论】研究建立的NIS1-PCR-RFLP标记系统可简便、直观地区分不同辣椒炭疽病菌的差异，有望发展成为真菌新的分子标记应用于田间辣椒炭疽病菌种群的快速鉴定。
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| 关 键 词： | 辣椒炭疽病菌 效应因子NIS1 PCR-RFLP 标记系统 核糖体DNA转录间隔区（rDNA-ITS） |
| 收稿时间： | 2021-01-14 |
Development of NIS1-PCR-RFLP marker system to effectors of Colletotrichum spp. from infected pepper plants |
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| OUYANG Chao,LIU Si-zhen,MAN Yi-long,SHENG Jia-wei,CHEN Yue,ZHANG Xin,LIU Yong,ZHANG De-yong,TAN Xin-qiu.Development of NIS1-PCR-RFLP marker system to effectors of Colletotrichum spp. from infected pepper plants[J].Journal of Southern Agriculture,2021,52(9):2473-2481. |
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| Authors: | OUYANG Chao LIU Si-zhen MAN Yi-long SHENG Jia-wei CHEN Yue ZHANG Xin LIU Yong ZHANG De-yong TAN Xin-qiu |
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| Affiliation: | 1 Long Ping Branch, Graduate School of Hunan University, Changsha 410125, China;2 Plant Protection Institute, Hunan Academy of Agricultural Sciences, Changsha 410125, China;3 Biotechnology Institute, Hunan Academy of Agricultural Sciences, Changsha 410125, China;4 Agricultural University of Hunan, Changsha 410000, China |
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| Abstract: | 【Objective】 Based on the complex population of pepper anthracnose and unclear composition of compound infection population in the fields,it was relatively difficult to control capsicum anthracis. Therefore,a rapid and intuitive marking system for pepper anthracnose was established to provide scientific basis for field control of the disease.【Method】 The rDNA-ITS primers were used to clone 22 isolates of pepper Colletotrichum spp. isolated from field. The phylogenetic tree was constructed by sequence alignment analysis,and the species and taxonomic status of 22 isolates were preliminarily determined. An effector NIS1 gene as target,its alignment was performed between Colletotrichum gloeosporioides and Colletotrichum orbiculare to design degenerate primers of NIS1 by genome database. NIS1 gene was isolated and cloned of 22 different strains of pepper Colletotrichum spp.,and phylogenetic tree of NIS1 gene was constructed with their NIS1 sequences alignment. Analyzed and compared the genes of rDNA-ITS and NIS1 gene to distinguish the difference of the tested strains,and then analyzed the NIS1 gene of 22 different strains of pepper Colletotrichum spp.,selected restriction endonuclease,analyzed the difference of PCR product restriction fragment length polymorphism(RFLP)of NIS1 gene,and established the NIS1-PCR-RFLP labeling system.【Result】 Phylogenetic analysis of rDNA-ITS sequences showed that 22 strains of Colletotrichum spp. belonged to 5 species of C. gloeosporioides,C. brevisporum,C. acutatum, C. truncatum and C. capsici,and their sequences homology was 85.6%-99.8%,but C. capsici and C. truncatum were in the same mixed group. The NIS1 gene sequences of 22 strains of Colletotrichum spp. were conserved,and there was variable region in the middle,which was 34.6%-78.9% homologous with the NIS1 gene of C. orbiculare. The NIS1 gene phylogenetic tree could divide 22 strains of Colletotrichum spp. into 5 groups,and C. capsici and C. truncatum were in different groups,indicating that their differentiation was better than rDNA-ITS. NIS1-PCR-RFLP showed that the maximum fragments of C. gloeosporioides and C. brevisporum were both 210 bp,but the number of fragments was different,while those of C. acutatum,C. capsici and C. truncatum were 292,685,and 345 bp,respectively. The reference strain C. orbiculare was 497 bp,which can be visually distinguished.【Conclusion】 The NIS1-PCR-RFLP marking system was established in this study,it can easily and directly distinguish the differences of different Colletotrichum spp. infected pepper plants,Therefore,the NIS1-PCR-RFLP marking system is expected to be developed into a new fungal molecular marker for rapid identification of Colletotrichum spp. from infected pepper plants in field. |
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