玉米大斑病菌ATMT突变体库的构建及其分析

doi:10.3864/j.issn.0578-1752.2012.12.006

摘要/Abstract

摘要： 【目的】利用农杆菌（Agrobacterium tumefaciens）介导的遗传转化技术，对玉米大斑病菌（Setosphaeria turcica）进行转化，构建ATMT突变体库，为从分子水平上揭示病菌的致病机理奠定基础。【方法】以带有重组双元载体的农杆菌对玉米大斑病菌进行转化，利用潮霉素B进行筛选，对抗性稳定的转化子进行PCR检测，构建玉米大斑病菌ATMT突变体库；从突变体库中随机选取一定数量的突变体，对其菌落形态、菌丝及分生孢子发育、致病性等进行分析。【结果】获得了1 265株玉米大斑病菌T-DNA插入突变体；从中随机选取36株突变菌株，对其进行抗性筛选和PCR检测，发现潮霉素磷酸转移酶基因已整合进入野生型菌株的基因组中，且能够稳定遗传；与野生型菌株相比，在供试的菌株中，大部分菌株菌落形态和生长速率没有发生明显改变。生长速率明显减慢的菌株占总数的13.8%，明显加快的菌株占16.7%；发现了2株分生孢子形态发生明显改变的菌株，占菌株总数的5.6%；产孢量明显增大的菌株占5.6%，产孢量减少的菌株占13.5%；分生孢子萌发率发生明显改变的菌株占16.6%；发现了1株致病性明显增强的菌株，占菌株总数的2.8%。【结论】构建了玉米大斑病菌ATMT突变体库，并对突变体库进行了初步分析，将为克隆玉米大斑病菌生长发育和致病性相关基因奠定基础。

关键词: 玉米大斑病菌, ATMT, 突变体库, T-DNA插入突变体

Abstract: 【Objective】 The objective of this study is to use Agrobacterium tumefaciens-mediated transformation (ATMT) to mutate the genes of Setosphaeria turcica and construct ATMT mutant library, which will lay a foundation for illuminating the pathogenesis of S. turcica at molecular level. 【Method】A. tumefacien with binary recombination vector was transformed into S. turcica, and the mutants were screened by hygromycin B and PCR technology. Based on the methods, a preliminary ATMT mutant library was constructed. Some mutants were selected randomly and analysed about the colony morphologies, mycelium and conidium development, and pathogenicity.【Result】A total of 1 265 T-DNA insertion mutants were obtained in this experiment. Hygromycin B resistance screening and PCR technology were used to confirm 36 mutants, which were selected randomly from the mutant library. The results showed that hygromycin phosphotransferase genes had been integrated into wild-type genome of S. turcica and the mutants were stable in their characteristics of genetics. Compared with wild-type strains, most of the mutants did not changed their colony morphologies and growth rates, only minorities of the mutants varied in their characteristics, in which 13.8% strains slowed down their growth rate obviously, 16.7% strains accelerated their growth rate distinctly, about 5.6% strains altered their shapes of conidia, 13.5% strains reduced the number of conidia, 16.6% strains changed their germination rate evidently, and 1 strain reinforced its pathogenicity, accounting 2.8% in all selected strains.【Conclusion】 ATMT mutant library of S. turcica was constructed and some mutants were analysed primarily. The study will lay a foundation for cloning genes related to growth, development and pathogenicity of S. turcica in the future.

Key words: Setosphaeria turcica, ATMT, mutant library, T-DNA insertion mutant

王梅娟, 李坡, 吴敏, 范永山, 谷守芹, 董金皋. 玉米大斑病菌ATMT突变体库的构建及其分析[J]. 中国农业科学, 2012, 45(12): 2384-2392.

WANG Mei-Juan, LI Po, WU Min, FAN Yong-Shan, GU Shou-Qin, DONG Jin-Gao. Constuction and Evaluation of ATMT Mutant Library of Setosphaeria turcica[J]. Scientia Agricultura Sinica, 2012, 45(12): 2384-2392.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2012.12.006

https://www.chinaagrisci.com/CN/Y2012/V45/I12/2384

参考文献

[1]Dong J G, Fan Y S, Gui X M, An X L, Ma J F, Dong Z P. Geographic distribution and genetic analysis of physiological races of Setosphaeria turcica in northern China. American Journal of Agricultural and Biological Sciences, 2008, 3(1): 389-398.

[2]Perkins J M, Pedersen W L. Disease development and yield losses associated with leaf northern blight on corn. Plant Disease, 1987, 71(10): 940-943.

[3]Rho H S, Kang S, Lee Y H. Agrobacterium tumefaciens-mediated transformation of the plant pathogenic fungus Magnaporthe grisea. Molecules and Cells, 2001, 12(3): 407-411.

[4]Mullins E D, Kang S. Transformation: a tool for studying fungal pathogens of plants. Cellular and Molecular Life Sciences, 2001, 58(14): 2043-2052.

[5]Michielse C B, Hooykaas P J J, Hondel C A M J J, Ram A F J. Agrobacterium-mediated transformation as a tool for functional genomics in fungi. Current Genetics, 2005, 48: 1-17.

[6]Bundock P, den Dulk-Ras A, Beijersbergen A, Hooykaas P J J. Trans-kingdom T-DNA transfer from Agrobacterium tumefaeiens to Saccharomyces cerevisiae. The EMBO Journal, 1995, 14(13): 3206-3214.

[7]de Groot M J A, Bundock P, Hooykass P J J, Beijersbergen A G M. Agrobacterium tumefaeiens mediated transformation of filamentous fungi. Nature Biotechnology, 1998, 16: 839-842.

[8]Mullins E D, Chen X, Romaine P, Raina R, Geiser D M, Kang S. Agrobacterium mediated transformation of Fusarium oxysporum: an efficient tool for insertional mutagenesis and gene transfer. Phytopathology, 2001, 91: 173-180.

[9]Covert S F, Kapoor P, Lee M H, Briley A, Nairn C J. Agrobaeterium tumefaeiens mediated transformation of Fusarium circinatum. Mycological Research, 2001, 105(3): 259-264.

[10]Choi J, Park J, Jeon J, Chi M H, Goh J, Yoo S Y, Park J, Jung K, Kim H, Park S Y, Rho H S, Kim S, Kim B R, Han S S, Kang S, Lee Y H. Genome-wide analysis of T-DNA integration into the chromosomes of Magnaporthe oryzae. Molecular Microbiology, 2007, 66(2): 371-382.

[11]Flowers J L, Vaillancourt L J. Parameters affecting the efficiency of Agrobacterium tumefaeiens-mediated transformation of Colletotrichum graminicola. Current Genetics, 2005, 48(6): 380-388.

[12]Sugui J A, Chang Y C, Kwon-Chung K J. Agrobacterium tumefaciens-mediated transformation of Aspergillus fumigatus: an efficient tool for insertional mutagenesis and targeted gene disruption. Applied and Environmental Microbiology, 2005, 71(4): 1798-1802.

[13]张震, 杜新法, 柴荣耀, 毛雪琴, 邱海萍, 王艳丽, 王教瑜, 孙国昌. 根癌农杆菌介导遗传转化稻曲病菌. 中国水稻科学, 2006, 20(4): 440- 442.

Zhang Z, Du X F, Chai R Y, Mao X Q, Qiu H P, Wang Y L, Wang J Y, Sun G C. Agrobacterium tumefaciens-mediated transformation of the pathogen of Ustilaginoidea virens. Chinese Journal of Rice Science, 2006, 20(4): 440-442. (in Chinese)

[14]王玉萍, 王晓鸣, 马青. 我国玉米大斑病菌生理小种组成变异研究. 玉米科学, 2007, 15(2): 123-126.

Wang Y P, Wang X M, Ma Q. Races of Exserohihun turcicum, causal agent of northern leaf blight in China. Journal of Maize Sciences, 2007, 15(2): 123-126. (in Chinese)

[15]安鑫龙, 郑晓莲, 董金皋. 玉米大斑病长蠕孢生理小种遗传变异. 微生物学通报, 2002, 29(2): 53-57.

An X L, Zheng X L, Dong J G. Genetic variation in races of Exserohihun turcicum. Microbiology, 2002, 29(2): 53-57. (in Chinese)

[16]Maruthachalam K, Klosterman S J, Kang S, Hayes R J, Subbarao K V. Identification of pathogenicity-related genes in the vascular wilt fungus Verticillium dahliae by Agrobacterium tumefaciens-mediated T-DNA insertional mutagenesis. Molecular Biotechnology, 2011, 49: 209-221.

[17]Combier J P, Melayah D, Raffier C, Gay G, Marmeisse R. Agrobacterium tumefaciens-mediated transformation as a tool for insertional mutagenesis in the symbiotic ectomycorrhizal fungus Hebeloma cylindrosporum. FEMS Microbiology Letters, 2003, 220: 141-148.

[18]刘建光, 王艳辉, 张国珍, 贾慧, 董金皋. 农杆菌介导的玉米大斑病菌的遗传转化. 植物病理学报, 2007, 37(6): 675-678.

Liu J G, Wang Y H, Zhang G Z, Jia H, Dong J G. Agrobacterium tumefaciens-mediated transformation of Setosphaeria turcica. Acta Phytopathologica Sinica, 2007, 37(6): 675-678. (in Chinese)

[19]张娇, 谷守芹, 李青为, 韩建民, 董金皋. 根癌农杆菌介导的玉米大斑病菌转化条件的优化. 河北农业大学学报, 2010, 33(4): 85-88, 108.

Zhang J, Gu S Q, Li Q W, Han J M, Dong J G. Optimization of factors affecting genetic transformation of Setosphaeria turcica via Agrobacterium tumefaciens. Journal of Agricultural University of Hebei, 2010, 33(4): 85-88, 108. (in Chinese)

[20]安鑫龙, 董金皋, 韩建民. 玉米大斑病菌的RAPD分析I. 应用CTAB法提取玉米大斑病菌DNA. 河北农业大学学报, 2001, 24(1): 38-41.

An X L, Dong J G, Han J M. RAPD analysis of Exserohilum turcicum I. CTAB method of isolating DNA from Exserohilum turcicum for RAPD. Journal of Agaricultural University of Hebei, 2001, 24(1): 38-41. (in Chinese)

[21]方丽, 刘海青, 宋凤鸣, 郑重. 农杆菌介导的黄瓜炭疽菌遗传转化. 浙江大学学报, 2006, 32(4): 360-366.

Fang L, Liu H Q, Song F M, Zheng Z. Agrobacterium tumefaciens- mediated transformation of Colletotrichum lagenarium, the causal agent of cucumber anthracnose. Journal of Zhejiang University, 2006, 32(4): 360-366. (in Chinese)

[22]冯娟, 朱廷恒, 崔志峰, 汪琨. 农杆菌介导的灰葡萄孢T-DNA插入突变体库构建及插入位点分析. 微生物学报, 2010, 50(2): 169-173.

Feng J, Zhu T H, Cui Z F, Wang K. Construction of T-DNA insertion mutants of Botrytis cinerea via Agrobacterium tumefaciens mediated transformation and sequence analysis of insertion site. Acta Microbiologica Sinica, 2010, 50(2): 169-173. (in Chinese)