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