一株拮抗香蕉枯萎病的内生细菌的分离及其几丁质酶基因信号肽的分泌活性分析

目的：旨在分离并选择一株香蕉内生细菌作为内生基因工程生防菌，并克隆其几丁质酶基因的信号肽序列。方法：从香蕉植株杆下部分离并选择了一株拮抗香蕉枯萎病且具有分泌几丁质酶能力的内生细菌，对该菌株进行了形态观察、生理生化测定和16S rDNA序列分析，克隆了其几丁质酶基因的编码序列并预测了其信号肽，构建了含有信号肽和不含信号肽的几丁质酶的表达菌株BL-chi1和BL-chi2。结果：结合形态观察、生理生化特征和16S rDNA序列比对分析确定该菌株为Klebsiella属，将该菌株命名为KKWB 5；BL-chi1和BL-chi2经IPTG诱导后，均表达了与预期蛋白大小一致的蛋白，同时BL-chi1诱导后的培养基上清中出现一条约45kDa的条带，而BL-chi2和空载体的BL-pET22b诱导后的培养基上清中均无此条带；几丁质水解试验发现，BL-chi1诱导后的培养基上清中的蛋白经浓缩和纯化后都能在几丁质平板上形成透明水解圈。结论：该几丁质酶的信号肽能被BL21（DE3）所识别，将几丁质酶分泌到培养基中，并且分泌的几丁质酶具有水解几丁质的生物学活性。内生菌KKWB-5的分离及其几丁质酶分泌信号肽序列的克隆为进一步构建内生工程菌来防治香蕉枯萎病打下了基础。

Isolation of a Strain of Endophytic Bacteria against Banana Fusarium Wilt and Activity Analysis of Signal Peptide of its Chitinase

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.