短小芽孢杆菌转座突变株的GFP标记及在水稻上的定殖

Labeling Bacillus pumillus with Green Fluorescent Protein (GFP) and Its Colonization in Rice Seedlings

【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。

【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.