山冈单胞菌ZL261几丁质酶基因的克隆及原核表达

Cloning and Prokaryotic Expression of the Chitinase Gene of Collimonas pratensis ZL261

分离自西藏色季拉山海拔4 530 m高山草甸土壤的山冈单胞菌(Collimonas pratensis)ZL261具有很高的几丁质酶活性,为了明确其几丁质酶种类及作用特点,依据已报道的食真菌山冈单胞菌(Collimonas fungivorans)Ter331胞外内切几丁质酶基因chiI相关序列设计引物,利用分段扩增的方法得到了菌株ZL261几丁质酶基因,并对其序列和编码蛋白的氨基酸进行分析。结果表明,全长1 338 bp的chiIQ基因编码的几丁质酶ChiIQ由445个氨基酸组成,分子量为46.04 kDa,等电点为5.67。序列比对和同源性分析发现,该基因与食真菌山冈单胞菌Ter331几丁质酶基因chiI序列相似性最高(92%),其氨基酸序列含有几丁质酶GH18家族高度保守序列SXGG和GXDXDXE,因此,ChiIQ属GH18家族。利用双酶切法构建了ZL261几丁质酶基因原核表达载体,并在大肠杆菌BL21中成功诱导表达。本试验结果为后期研究几丁质酶在山冈单胞菌生防应用中的功能以及生防菌ZL261的开发和应用奠定了基础。

Collimonas prokaryotic ZL261 was isolated from the alpine meadow soil at Sejila Mountain with 4530 meters altitude in Tibet of China. The strain was able to produce a high active chitinase which formed large transparent zones on chitinase testing medium plates. In order to clarify the category and the function characteristics of the chitinase, primers designed based on the extracellular chitinase gene chiI of C. fungivorans Ter331 were used to clone the chitinase gene in strain ZL261. The results showed that the chitinase ChilQ is 1338 bp in length and consists of 445 amino acids with a molecular weight of 46.04 kDa and a predicted isoelectric point of 5.67. Sequence alignment and homology analysis revealed that the chilQ gene had a high similarity （92%） to the chil gene of the strain Ter331. The amino acid sequence of ChiIQ contained the highly conserved SXGG and GXDXDXE domains of chitinase GH18 family. Therefore, ChiIQ chitinase was a member of GH18 family. The prokaryotic expression vector of chilQ was constructed using restriction endonucleases digestion method and expressed in E. coli BL21 successfully. The results provided the important information for application of chitinase, and the development and application of strain ZL261.