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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16SrDNA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16SrDNA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。
OBJECTIVE: To isolate and select a banana endophytic bacteria as an endogenous genetic engineering antimicrobial, and clone the signal peptide sequence of its chitinase gene. METHODS: An endophytic bacterium that antagonized banana wilt disease and secreted chitinase was isolated and isolated from the lower part of banana plant. Morphological, physiological and biochemical analyzes and 16S rDNA sequence analysis of the strain were carried out. The coding sequence of the enzyme gene was cloned and its signal peptide was predicted. The expression strains BL-chi1 and BL-chi2 containing signal peptide and chitinase without signal peptide were constructed. Results: The strain was identified as KKWB-5 by morphological observation, physiological and biochemical characteristics and 16S rDNA sequence analysis. After induced by IPTG, BL-chi1 and BL-chi2 expressed the same protein A protein of about 45kDa appeared in the supernatant of the medium after induction by BL-chi1, and no such band was observed in the medium supernatant of BL-chi2 and empty vector after induction by BL-pET22b; The results of the hydrolysis of butyryl showed that the protein in the supernatant of the medium after BL-chi1 induction could form a clear hydrolysis circle on the chitin plate after concentration and purification. Conclusion: The chitinase signal peptide can be recognized by BL21 (DE3), secreting chitinase into the medium, and the secreted chitinase has the biological activity of hydrolyzing chitin. The isolation of endophyte KKWB-5 and the cloning of its chitinase secretion signal peptide sequence laid the foundation for the further construction of endogenous engineering bacteria to prevent and cure banana wilt disease.