短小芽孢杆菌转座突变株的GFP标记及在水稻上的定殖

doi:10.3864/j.issn.0578-1752.2012.24.007

摘要/Abstract

摘要： 【目的】研究GFP标记的短小芽孢杆菌在水稻植株上的定殖规律，为其生物防治应用提供科学依据。【方法】从短小芽孢杆菌GFP标记的转座突变株库中筛选强表达GFP且遗传稳定的突变株，用作水稻植株的定殖示踪。【结果】采用荧光酶标仪、流式细胞术检测及荧光显微镜观察等手段，对1 467株Tn5-egfp随机插入突变株的GFP表达作了定量和定性分析，最终获得8株荧光表达较强的突变株，并对突变株作了T-DNA插入拷贝数鉴定。在开放和封闭系统中，标记菌在水稻幼苗根际土壤中均可以存活15 d以上，且前者标记菌的数量下降相对更慢。【结论】短小芽孢杆菌在根毛区及侧根分生处的数量最多，并在根表面形成菌膜。该菌可通过水稻幼苗根部的伤口或幼嫩根毛等部位侵入根系，主要分布在皮层细胞及其间隙，在植物的维管束内亦可观察到荧光标记菌。研究结果表明该菌在水稻幼苗根际土壤中有着良好的定殖能力。

关键词: 水稻 , 短小芽孢杆菌 , Tn5转座 , 绿色荧光蛋白 , 定殖

Abstract: 【Objective】 The objective of this study is to investigate bacterial colonization in rice seedlings by GFP-labeled Bacillus pumilus DX01 and lay a solid foundation for the application of phytopathogenic biocontrol. 【Method】 Genetically stable mutants of B. pumilus DX01 with a strong GFP expression were identified from the GFP-labeled Tn5 insertion library and used for tracing the bacterial colonization in rice seedlings. 【Result】 A total of 1 467 mutants were subjected to qualitative and quantitative analyses of GFP expression by using fluorescence microplate reader, fluorescence activating cell sorter and fluorescent microscopy, and eventually 8 mutants with significantly enhanced GFP expression were identified. Meanwhile, the copy numbers of exogenous DNA integration in the genome of these mutants were detected. The GFP-tagged bacterial cells that released into the rhizosphere of rice seedlings could survive for more than 15 days. Furthermore, decrease of the B. pumilus bacterial cells in undisinfected soils showed more slowly compared with corresponding sterile soils. 【Conclusion】 B. pumilus mainly locates in root hair region and later root branch. Meanwhile, the bacterial film formed on rice root surface was also confirmed. B. pumilus can invade plant through the wound of roots or by young root hairs, and they distribute mostly in root cortex cells and intercellular spaces of cortical layer. The marked bacteria were often found in the vascular bundle of rice seedlings. In conclusion, B. pumilus displayed a good colonization ability in the rhizosphere soils of rice seedlings.

Key words: Oryza sativa , Bacillus pumillus , Tn5 transposition , green fluorescent protein , colonization

沈新迁, 刘通, 胡晓璐, 顾振芳, 陈云鹏. 短小芽孢杆菌转座突变株的GFP标记及在水稻上的定殖[J]. 中国农业科学, 2012, 45(24): 5024-5031.

SHEN Xin-Qian, LIU Tong, HU Xiao-Lu, GU Zhen-Fang, CHEN Yun-Peng. Labeling Bacillus pumillus with Green Fluorescent Protein (GFP) and Its Colonization in Rice Seedlings[J]. Scientia Agricultura Sinica, 2012, 45(24): 5024-5031.

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参考文献

[1]Bottone E J, Peluso R W. Production by Bacillus pumilus (MSH) of an antifungal compound that is active against Mucoraceae and Aspergillus species: Preliminary report. Journal of Medical Microbiology, 2003, 52(1): 69-74.

[2]Sun X B, Chen Y P, Wu C R, Yang G X, Guo B, Shen D L. Functional evaluation of a novel constitutive promoter F1 of Bacillus pumilus, as a rice epiphytic strain, and construction of an efficient expression and secretion system under the control of F1. Biotechnology Letters, 2006, 28(13): 979-985.

[3]Danhorn T, Fuqua C. Biofilm formation by plant-associated bacteria. Annual Review of Microbiology, 2007, 61: 401-422.

[4]Trifonova R, Postma J, Schilder M T, van Elsas J D. Microbial enrichment of a novel growing substrate and its effect on plant growth. Microbial Ecology, 2009, 58(3): 632-641.

[5]Raupach G S, Kloepper J W. Mixtures of plant growth-promoting rhizobacteria enhance biological control of multiple cucumber pathogens. Phytopathology, 1998, 88(11): 1158-1164.

[6]Berg G. Plant-microbe interactions promoting plant growth and health: perspective for controlled use of microorganisms in agriculture. Applied Microbiology and Biotechnology, 2009, 84: 11-18.

[7]March J C, Rao G, Bentley W E. Biotechnological applications of green fluorescent protein. Applied Microbiology and Biotechnology, 2003, 62(4): 303-315.

[8]Larrainzar E, O'Gara F, Morrissey J P. Applications of autofluorescent proteins for in situ studies in microbial ecology. Annual Review of Microbiology, 2005, 59: 257-277.

[9]Pinheiro L B, Gibbs M D, Vesey G, Smith J J, Bergquist P L. Fluorescent reference strains of bacteria by chromosomal integration of a modified green fluorescent protein gene. Applied Microbiology and Biotechnology, 2008, 77(6): 1287-1295.

[10]Kaltwasser M, Wiegert T, Schumann W. Construction and application of epitope- and green fluorescent protein-tagging integration vectors for Bacillus subtilis. Applied and Environmental Microbiology, 2002, 68(5): 2624-2628.

[11]Chen Y P, Yan J, Yang M J, Wang J W, Shen D L. Expression of green fluorescent protein in Bacillus brevis under the control of a novel constitutive promoter F1 and insertion mutagenesis of F1 in Escherichia coli DH5α. FEMS Microbiology Letters, 2003, 229(1): 111-117.

[12]Timmusk S, Grantcharova N, Wagner E G H. Paenibacillus polymyxa invades plant roots and forms biofilms. Applied and Environmental Microbiology, 2005, 71(11): 7292-7300.

[13]Olubajo B, Bacon C W. Electrotransformation of Bacillus mojavensis with fluorescent protein markers. Journal of Microbiological Methods, 2008, 74(2/3): 102-105.

[14]Fan B, Chen X H, Budiharjo A, Bleiss W, Vater J, Borriss R. Efficient colonization of plant roots by the plant growth promoting bacterium Bacillus amyloliquefaciens FZB42, engineered to express green fluorescent protein. Journal of Biotechnology, 2011, 151(4): 303-311.

[15]Ito M, Kim Y G, Tsuji H, Kiwaki M, Nomoto K, Tanaka R, Okada N, Danbara H. A practical random mutagenesis system for probiotic Lactobacillus casei using Tn5 transposition complexes. Journal of Applied Microbiology, 2010, 109(2): 657-666.

[16]Sant'Anna F H, Andrade D S, Trentini D B, Weber S S, Schrank I S. Tools for genetic manipulation of the plant growth-promoting bacterium Azospirillum amazonense. BMC Microbiology, 2011, 11: 107-115.

[17]沈新迁, 胡晓璐, 刘通, 孙文良, 陈云鹏. GFP标记的短小芽孢杆菌 (Bacillus pumilus) 转座突变株的构建初探. 上海交通大学学报: 农业科学版, 2012, 30(4): 15-20.

Shen X Q, Hu X L, Liu T, Sun W L, Chen Y P. Construction of GFP-labeled Tn5 insertion mutants of Bacillus pumilus and gfp expression analysis. Journal of Shanghai Jiaotong University: Agricultural Science, 2012, 30(4): 15-20. (in Chinese)

[18]Golan A, Kerem Z, Tun O M, Luzzatto T, Lipsky A, Yedidia I. Combining flow cytometry and gfp reporter gene for quantitative evaluation of Pectpbacterium carotovorum ssp. carotovorum in Ornithogalum dubium plantlets. Journal of Applied Microbiology, 2010, 108(4): 1136-1144.

[19]Han S W, Park C J, Lee S W, Ronald P C. An efficient method for visualization and growth of fluorescent Xanthomonas oryzae pv. oryzae in planta. BMC Microbiology, 2008, 8: 164-173.

[20]Unge A, Tombolini R, Molbak L, Jansson J K. Simultaneous monitoring of cell number and metabolic activity of specific bacterial populations with a dualgfp-luxAB marker system. Applied and Environmental Microbiology, 1999, 65(2): 813-821.

[21]Lamb T G, Tonkyn D W, Kluepfel D A. Movement of Pseudomonas aureofaciens from the rhizosphere to aerial plant tissue. Canadian Journal of Microbiology, 1996, 42(11): 1112-1120.

[22]Miao Y X, Zhou J, Chen C C, Shen D L, Song W, Feng Y J. In vitro adsorption revealing an apparent strong interaction between endophyte Pantoea agglomerans YS19 and host rice. Current Microbiology, 2008, 57(6): 547-551.

[23]Zhang X, Li E, Xiong X L, Shen D L, Feng Y J. Colonization of endophyte Pantoea agglomerans YS19 on host rice, with formation of multicellular symplasmata. World Journal of Microbiology and Biotechnology, 2010, 26 (9): 1667-1673.