内生芽孢杆菌BS2的β-1,4-内切葡聚糖酶基因与定殖相关性

doi:10.3864/j.issn.0578-1752.2014.02.006

摘要/Abstract

摘要： 【目的】克隆植物内生解淀粉芽孢杆菌（Bacillus amyloliquefaciens）BS2的β-1,4-内切葡聚糖酶基因（β-1,4-endoglucanase gene，eglS），探明BS2中该基因与定殖的相关性。【方法】以枯草芽孢杆菌BS168基因组为模板扩增组成型启动子rpsD。依据芽孢杆菌β-1,4-内切葡聚糖酶基因的同源序列设计1对引物，以BS2基因组为模板扩增eglS。将rpsD启动子基因和eglS片段用T4连接酶连接，并对连接产物进行PCR扩增，得到eglS表达盒片段。将表达盒片段插入穿梭载体pGFP4412用以构建eglS过表达质粒。设计引物扩增eglS的同源重组片段，与具有单交换功能的整合载体pMUTIN-GFP+连接构建敲除质粒。eglS的过表达质粒和敲除质粒转化芽孢杆菌感受态细胞分别获得过表达突变体和敲除突变体。将过表达质粒转化敲除突变体感受态细胞获得互补突变体。采用荧光显微镜观察、PCR、平板酶活力检测及实时荧光定量PCR方法鉴定突变体。3,5-二硝基水杨酸比色法（DNS法）测定突变体和野生菌的β-1,4-内切葡聚糖酶活力，同时采用定量灌根接种法检测不同菌株30 d内在小白菜各组织中的定殖数量。【结果】获得在荧光显微镜下呈现绿色荧光的过表达突变体和互补突变体。PCR检测结果表明敲除突变体中能够扩增出675 bp的目的条带，而以野生菌株和整合载体DNA为模板扩增时没有对应条带，成功得到敲除突变体。野生菌及各突变体β-1,4-内切葡聚糖酶平板活力检测结果显示，野生菌BS2只有一个水解圈且透明，过表达突变体与互补突变体有大于野生菌的双水解圈，内圈的水解圈透明，外圈的水解圈不透明。敲除突变体沿菌体边缘也有一个小水解圈。实时荧光定量PCR的分析结果证实野生菌及突变体间β-1,4-内切葡聚糖酶基因的表达量存在差异，过表达突变体和互补突变体中β-1,4-内切葡聚糖酶基因的表达量增高，分别是野生菌的111和82倍；敲除突变体中β-1,4-内切葡聚糖酶基因的表达量相对于野生菌BS2几乎为0。突变体与野生菌在小白菜体内定殖数量以及酶活力检测结果表明，在相同的小白菜组织以及相同的分离时间内，敲除突变体的定殖数量最少，其在根茎叶中的定殖数量与其他菌株相比均有显著性差异（P＜0.05）。敲除突变体的酶活力也显著低于其他菌株（P＜0.05）。过表达突变体的酶活力以及在小白菜体内的定殖数量在P＜0.05水平上显著高于敲除突变体和野生菌，互补突变体的酶活力和在小白菜体内的定殖数量与过表达突变体基本一致。【结论】内生芽孢杆菌BS2菌株β-1,4-内切葡聚糖酶的活力高低与其在小白菜体内的定殖数量之间呈正相关关系，其表达的β-1,4-内切葡聚糖酶在定殖过程中发挥一定作用。

关键词: 内生芽孢杆菌BS2 , &beta, -1 , 4-内切葡聚糖酶 , 过表达 , 基因敲除 , 定殖

Abstract: 【Objective】 The objective of this study is to clone β-1,4-endoglucanase gene (eglS) of endophytic Bacillus amyloliquefaciens strain BS2, investigate the interactive relationship between the gene and plant in colonization. 【Method】The constitutive promoter rpsD sequence was cloned from genomic DNA of B. subtilis 168 by PCR. Homology primers were used to clone eglS from genomic DNA of strain BS2. Then the fragments of rpsD and eglS were ligated and amplified by PCR, and an expression cassette was obtained. The expression cassette fragment was inserted into the shuttle vector pGFP4412 for constructing over-expressive plasmid. pGFP4412 was digested by EcoR I and Xba I. PCR was also used to obtain homologous recombination fragment of eglS. The homologous recombination fragment was inserted into the integration vector pMUTIN-GFP+, which could integrate into chromosome by a single recombination event, to construct knockout plasmid. The over-expressive mutants and knockout mutants were obtained by competent-cell transformation of over-expression plasmids and knockout plasmids, respectively. Complementary mutants were generated by transforming over-expressive plasmids into knockout mutants. The mutants were confirmed by a fluorescence microscope, PCR, enzymatic assays on agar plates and real-time PCR. Enzyme activity of β-1,4-endoglucanase was determined by 3,5-dinitrosalicylic acid colorimetric method (DNS method). The colonizing bacteria amounts of mutants and the wild strain in the tissues of cabbage plants within 30 d were measured after inoculation by root irrigation. 【Result】The over-expressive mutants and complementary mutants were obtained, which showed green fluorescence under a fluorescent microscope. Knockout mutants were identified by PCR and the results showed that the DNA fragment of about 675 bp was amplified from the knockout mutants. However, there were no corresponding bands from the wild-type strain and the integration vector. β-1,4-endoglucanase assays on agar plates showed that the over-expression mutants and complementary mutants presented large and dual hydrolytic circles compared with the wild-type strain. In addition, the clarity of the outer circle was less than that of the inner one. The knockout mutants showed a small and very weak hydrolytic circle around the edge of a colony. The real-time quantitative PCR results confirmed the differentially expression of β-1,4-endoglucanase between the wild-type strain and the mutants. The relative mRNA transcription levels of β-1,4-endoglucanase gene in the over-expressive mutants and complementary mutants were higher than that in the wild-type strain. They were 111 and 82 times as much as that of the wild-type strain, respectively. In contrast, the relative expression level of β-1,4-endoglucanase mRNA in knockout mutants was approximately 0 compared with the wild-type strain. The results of quantity of mutants and the wild strain in the tissues of cabbage plants and enzyme activity assay showed that knockout mutant bacteria had the least amount of number in the same cabbage plant tissues and the same isolation time, and it had significant difference compared with other strains (P<0.05). In addition, the β-1,4-endoglucanase activity of knockout mutants was also the lowest, and it was significantly lower than that in the other strains (P<0.05). The over-expression mutants were significantly higher than that of the knockout mutants and wild strains both in enzyme activity and colonization population in the cabbage plants (P<0.05). The enzyme activity and colonizing bacteria density in the cabbage plants of complementary mutants were basically consistent with the over-expression mutants.【Conclusion】There was a positive correlation between the level of β-1,4-endoglucanase activity and its colonization quantity in the cabbage plants of the endophytic B. amyloliquefaciens BS2. The β-1,4-endoglucanase of strain BS2 played a role in the process of colonization in plants.

Key words: endophytic Bacillus amyloliquefaciens BS2 , β-1 , 4-endoglucanase , over-expression , gene knockout , colonization

范晓静1, 杨瑞先1, 2, 邱思鑫3, 胡方平1. 内生芽孢杆菌BS2的β-1,4-内切葡聚糖酶基因与定殖相关性[J]. 中国农业科学, 2014, 47(2): 262-272.

FAN Xiao-Jing-1, YANG Rui-Xian-1, 2 , QIU Si-Xin-3, HU Fang-Ping-1. Relationship Between β-1, 4-Endoglucanase Gene in Endophytic Bacillus Strain BS2 and Its Colonization in Plant[J]. Scientia Agricultura Sinica, 2014, 47(2): 262-272.

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