山冈单胞菌ZL261几丁质酶基因的克隆及原核表达

发布时间：2018-07-05 07:14

本文选题：山冈单胞菌 + 几丁质酶　；参考：《核农学报》2017年11期

【摘要】：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。
[Abstract]:Collimonas pratensis ZL261 isolated from the alpine meadow soil of Serjila Mountain, Tibet, has high chitinase activity. Based on the reported sequence of chitinase gene of Collimonas fungivorans Ter331, primers were designed. Strain ZL261 chitinase gene was obtained by segmental amplification, and its sequence and amino acid encoding protein were analyzed. The results showed that chiIQ gene encoding chitinase ChiIQ was composed of 445 amino acids with molecular weight of 46.04 kDa and isoelectric point of 5.67. Sequence alignment and homology analysis showed that this gene had the highest similarity (92%) with the chiI sequence of Ter331 chitinase gene of fungi Ter331. Its amino acid sequence contained highly conserved sequences of chitinase GH18 family SXGG and GXDXDXE, so ChiIQ belongs to GH18 family. The prokaryotic expression vector of ZL261 chitinase gene was constructed by double enzyme digestion and successfully induced in Escherichia coli BL21. The results laid a foundation for studying the function of chitinase in biocontrol and the development and application of biocontrol bacteria ZL261.
【作者单位】：北京市农林科学院植物保护环境保护研究所;广东海洋大学农学院;
【基金】：北京市科技计划课题(D151100003915003) 北京市优秀人才青年骨干个人项目(2014000020060G180) 北京市农林科学院科技创新能力建设专项(KJCX20170410)
【分类号】：Q78;Q93

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