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  

Relationship Between β-1,4-Endoglucanase Gene in Endophytic Bacillus Strain BS2 and Its Colonization in Plant

作　　者：范晓静[1] 杨瑞先 邱思鑫[3] 胡方平[1] 

机构地区：[1]福建农林大学植物保护学院,福州350002 [2]洛阳理工学院环境工程与化学系,河南洛阳471023 [3]福建省农业科学院作物研究所,福州350013

出　　处：《中国农业科学》2014年第2期262-272,共11页Scientia Agricultura Sinica

基　　金：福建省科技厅自然科学基金项目(2010J05049)

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;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

关 键 词：内生芽孢杆菌BS2 Β-1 4-内切葡聚糖酶 过表达 基因敲除 定殖 

分 类 号：S476[农业科学—农业昆虫与害虫防治]