L-脯氨酸对枯草芽胞杆菌NCD-2菌株生物膜形成的影响  

Effect of L-proline on biofilm formation of Bacillus subtilis NCD-2

作　　者：赵卫松[1] 郭庆港[1] 董丽红[1] 王培培[1] 张晓云[1] 苏振贺 鹿秀云[1] 李社增[1] 马平[1] ZHAO Wei-song;GUO Qing-gang;DONG Li-hong;WANG Pei-pei;ZHANG Xiao-yun;SU Zhen-he;LU Xiu-yun;LI She-zeng;MA Ping(Plant Protection Institute of Hebei Academy of Agricultural and Forestry Sciences/IPM Centre of Hebei Province/Key Laboratory of IPM on Crops in Northern Region of North China,Ministry of Agriculture and Rural Affairs,Baoding 071000,China)

机构地区：[1]河北省农林科学院植物保护研究所/河北省农业有害生物综合防治工程技术研究中心/农业农村部华北北部作物有害生物综合治理重点实验室,保定071000

出　　处：《植物病理学报》2021年第1期115-122,共8页Acta Phytopathologica Sinica

基　　金：国家自然科学基金(31801786、31601680);河北省农林科学院科学技术研究与发展计划(201820101);国家棉花产业技术体系(CARS-15-17)。

摘　　要：枯草芽胞杆菌NCD-2菌株在棉花品种‘冀棉11’根际的定殖能力与其根系分泌物中的L-脯氨酸相关。本研究通过外源添加L-脯氨酸评价其对NCD-2菌株生长量和生物膜形成的影响。结果表明,在MSgg培养基中添加L-脯氨酸培养48 h后对菌株生长不存在显著影响。采用称重法测定生物膜的湿重和干重,结果表明在MSgg培养基添加L-脯氨酸显著增加了生物膜产量,且生物膜的褶皱程度和立体结构变得更为明显。采用结晶紫染色法进行生物膜的定量测定发现,浓度为1.250~10.000 mg·mL^(-1)的L-脯氨酸可以显著提高菌株生物膜形成能力,而低浓度0.625 mg·mL^(-1)的L-脯氨酸对生物膜形成无影响。通过RT-qPCR检测了NCD-2菌株生物膜形成相关基因tasA和epsA表达量的变化,结果表明,添加上述不同浓度L-脯氨酸能够显著提高tasA基因表达量,提高了2.53~9.98倍。除了浓度为0.625 mg·mL^(-1) L-脯氨酸可提高epsA基因表达外,其他浓度没有显著改变epsA基因的表达,而且epsA基因表达量低于tasA基因。综合分析表明,L-脯氨酸促进NCD-2菌株生物膜形成不是通过改变菌株生长量,而是诱导tasA基因的表达产生更多的胞外蛋白TasA。The colonization ability of Bacillus subtilis NCD-2 in the rhizosphere of ’Jimian 11’ is associated with L-proline in its root exudates.The effect of exogenous L-proline on the growth and biofilm formation of strain NCD-2 was determined in this study.The result showed that addition of L-proline to the MSgg medium did not affect on the growth of strain NCD-2,while the yield of biofilm was increased significantly and the degree of wrinkling and stereo-structure of biofilm were more obvious.The quantitative determination of biofilm formation was performed by crystal violet staining.The results showed that a higher concentration of L-proline at 1.250 to10.000 mg·mL^(-1)(but not 0.625 mg·mL^(-1)) could increase biofilm formation of strain NCD-2 compared to the mock control.The expression levels of tasA and epsA genes,two biofilm formation-related markers were analyzed by RT-qPCR after treatment with L-proline.Expression of the tasA gene was significantly increased by2.53 to 9.98 times at different concentrations of L-proline.However,expression of the epsA gene was only increased in 0.625 mg·mL^(-1) L-proline,and the expression level of the epsA gene was lower than that of tasA gene.These results suggest that supply of L-proline enhances biofilm formation of B.subtilis NCD-2 through boosting the production of extracellular protein TasA rather than increasing bacterial growth.

关 键 词：枯草芽胞杆菌 L-脯氨酸 生物膜形成 定殖 RT-QPCR 

分 类 号：S476[农业科学—农业昆虫与害虫防治]