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| Isolation of a Strain of Endophytic Bacteria against Banana Fusarium Wilt and Activity Analysis of Signal Peptide of its Chitinase |
| XIA Qi-yu,WANG YU-guang,SUN Jian-bo,LU Xue-hua,GU Wen-liang |
| Key Laboratory of Tropical Crop Biotechnology, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101,China
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Abstract To construct an endophytic engineered bacterium, a strain of endophytic bacteria with resistance to banana Fusarium wilt and ability to degrade chitin was isolated from healthy banana plants. This strain was identified as Klebsiella based on its morphological, physiological and biochemical characteristics and 16S rDNA sequences analysis. The strain was named as KKWB-5. In order to obtain a secretion signal peptide, the chitinase II gene of KKWB-5 was cloned by PCR, and its signal peptide sequence was forecasted by Signal P3.0 Server. The chitinase gene was inserted into E.coli BL21(DE3) by pET22b to construct respectively the expression strain BL-chi1 and BL-chi2. The chitinase gene of BL-chi1 included the signal peptide sequence, while BL-chi2 removed it. After induced with IPTG, both BL-chi1 and BL-chi2 expressed a protein about 45KDa. The culture supernatant of BL-chi1 had a protein band about 45KDa too, while the culture supernatant of BL-chi2 and BL-pET22b control didn’t have. Biological activity determination showed that both concentrated liquid and purified liquid of the culture supernatant of BL-chi1 induced formed a clearing zone on the colloidal chitin plate. The results demonstrated the signal peptide sequence could be identified by E.coli BL21(DE3) and secrete the chitinase into culture supernatant, moreover, the secreted chitinase had biological activity. The isolation of KKWB-5 strain and cloning of signal peptide sequence of its chitinase served as a good foundation for further constructing engineered endophytic bacterium.
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Received: 23 April 2010
Published: 25 August 2010
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