利用绿色荧光蛋白报告基因标记研究麦根腐平脐孺孢对小麦根和叶片的侵染

doi:10.3864/j.issn.0578-1752.2012.17.006

中国农业科学 ›› 2012, Vol. 45 ›› Issue (17): 3506-3514.doi: 10.3864/j.issn.0578-1752.2012.17.006

利用绿色荧光蛋白报告基因标记研究麦根腐平脐孺孢对小麦根和叶片的侵染

闫立敏, 王晓鸣, 徐荣旗, 东方阳, 李洪杰

1.河北科技师范学院生命科技学院，河北秦皇岛066004
2.中国农业科学院作物科学研究所/农作物基因资源与基因改良国家重大科学工程，北京100081
3.中国农业科学院生物技术研究所，北京 100081

收稿日期:2011-12-29 出版日期:2012-09-01 发布日期:2012-03-05
通讯作者: 通信作者李洪杰，Tel：010-82109609；E-mail：hongjie@caas.net.cn；通信作者东方阳，Tel：0335-8076536；E-mail：yang_dogfang@hotmail.com
作者简介:闫立敏，Tel：010-82109609；E-mail：yanlimin2009@163.com
基金资助:
国家小麦产业技术体系专项（CARS-3-1）、国家转基因生物新品种培育科技重大专项（2009ZX08002-006B）

Root and Leaf Infection as Revealed by Autofluorescent Reporter Protein GFP Labeled Bipolaris sorokiniana in Wheat

YAN Li-Min, WANG Xiao-Ming, XU Rong-Qi, DONG Fang-Yang, LI Hong-Jie

1.河北科技师范学院生命科技学院，河北秦皇岛066004
2.中国农业科学院作物科学研究所/农作物基因资源与基因改良国家重大科学工程，北京100081
3.中国农业科学院生物技术研究所，北京 100081

Received:2011-12-29 Online:2012-09-01 Published:2012-03-05

摘要/Abstract

摘要： 【目的】利用绿色荧光蛋白标记研究麦根腐平脐孺孢（Bipolaris sorokiniana，引起小麦根腐病和叶枯病）对小麦根系和叶片的侵染过程，建立直观、非破坏性研究病原菌—植物互作的体系。【方法】采用农杆菌介导法将gfp导入麦根腐平脐孺孢菌株Bs-1中，对转化子进行荧光表达、PCR验证、遗传稳定性、生长特性和胞外酶代谢分析。选用与野生型菌株表现相近的转化子Bs-GFP研究麦根腐平脐孺孢对小麦品种矮抗58根和叶片的侵染过程。【结果】转化子Bs-GFP的菌丝和分生孢子表达明亮的绿色荧光，利用基因特异标记扩增证明gfp被整合到真菌的基因组中。对GFP标记菌株Bs-GFP的遗传稳定性和生长特性分析表明，gfp在转化子中能稳定地遗传，菌落的生长速度和胞外酶代谢与野生型菌株相比没有显著差异。菌株Bs-GFP可以在小麦的地下和地上部分引起病症，而且与野生型菌株Bs-1在小麦植株根系和茎基部组织定殖的数量（即CFU值）相近。【结论】利用农杆菌介导方法获得表达绿色荧光的麦根腐平脐孺孢菌株可以直观地观察病原菌对寄主的侵染过程，研究结果有助于更好地解析麦根腐平脐孺孢与小麦以及其它禾谷类寄主之间的互作。

关键词: 农杆菌, 麦根腐平脐孺孢, GFP, 遗传转化

Abstract: 【Objective】The objective of this study is to label Bipolaris sorokiniana, which is the causal agent of common root rot and leaf spot blotch of wheat (Triticum aestivum L.), with green fluorescent protein (GFP) for studying infection of B. sorokiniana on wheat roots and leaves, and to establish a direct and non-destructive method for in vivo observing interaction between pathogen and host plant. 【Method】 Gene gfp was transferred into B. sorokiniana strain Bs-1 via Agrobacterium tumefaciens-mediated transformation (ATMT). Based on analysis of fluorescence expression, PCR verification, genetic stability, growth parameters, and metabolism of extra cellular enzymes, a transformed strain Bs-GFP that resembled its wild-type strain, was chosen to investigate infection of B. sorokiniana on roots and leaves of wheat cultivar Aikang 58. 【Result】Bright green fluorescence was observed in hyphae and conidia of the transformant Bs-GFP. The amplification of the diagnostic bands with the gene-specific markers indicated that gfp was integrated into the genome of B. sorokiniana. Analysis of genetic stability and growth parameters demonstrated that gfp was inherited normally, and the growth rate and metabolism of extracellular enzymes were comparable to the wild type strain. The GFP-labeled strain Bs-GFP was able to incite symptoms on both underground and upperground parts of wheat plants, and the quantities of colonization (in terms of colony forming unit) in roots and basal stem tissues of wheat plants by Bs-GFP was similar as those by Bs-1. 【Conclusion】The B. sorokiniana strain expressing GFP obtained via ATMT can be used to directly monitor infection and colonization of the fungus on wheat. The results from this study will facilitate better understanding of the interactions between B. sorokiniana and wheat, as well other cereal crops.

Key words: Agrobacterium tumefaciens, Bipolaris sorokiniana (teleomorph Cochliobolus sativus), GFP (green fluorescence protein), genetic transformation

闫立敏, 王晓鸣, 徐荣旗, 东方阳, 李洪杰. 利用绿色荧光蛋白报告基因标记研究麦根腐平脐孺孢对小麦根和叶片的侵染[J]. 中国农业科学, 2012, 45(17): 3506-3514.

YAN Li-Min, WANG Xiao-Ming, XU Rong-Qi, DONG Fang-Yang, LI Hong-Jie. Root and Leaf Infection as Revealed by Autofluorescent Reporter Protein GFP Labeled Bipolaris sorokiniana in Wheat[J]. Scientia Agricultura Sinica, 2012, 45(17): 3506-3514.

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利用绿色荧光蛋白报告基因标记研究麦根腐平脐孺孢对小麦根和叶片的侵染

Root and Leaf Infection as Revealed by Autofluorescent Reporter Protein GFP Labeled Bipolaris sorokiniana in Wheat

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