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当前位置: 首页 > 文章 > 绿色木霉几丁质酶基因Tvchi cDNA的克隆、原核表达与活性分析 植物病理学报 2012,42 (2) 139-145
Position: Home > Articles > Cloning,prokaryotic expression and activity assay of chitinase(Tvchi) from Trichoderma viride Acta Phytopathologica Sinica 2012,42 (2) 139-145

绿色木霉几丁质酶基因Tvchi cDNA的克隆、原核表达与活性分析

作  者：
杨丽荣;孙虎;雷振生;赵献林;全鑫;薛保国
单  位：
河南省农业科学院小麦研究中心;河南省农业科学院植物保护研究所/河南省农作物病虫害防治重点实验室/农业部华北南部作物有害生物综合治理重点实验室
关键词：
绿色木霉;几丁质酶基因;基因克隆;原核表达
摘  要：
利用RT-PCR方法从绿木霉ZBS-6中扩增了几丁质酶基因Tvchi,序列分析表明Tvchi开放阅读框为1 293 bp,编码430个氨基酸残基,Blast分析表明,与多种木霉18家族内切几丁质酶具有较高的同源性。将该基因克隆到原核表达载体pET-28a上,转化大肠杆菌BL21(DE3),经IPTG诱导,SDS-PAGE和Western印迹分析表明成功的获得了47 kD的融合蛋白。该融合蛋白经Ni-NTA柱亲和纯化,获得了纯度较高的融合蛋白Tvchi。Tvchi最适酶活温度为37℃,最适酶活pH值为6.8。表达产物对小麦全蚀病、赤霉病、纹枯病病原菌显示出较好的抑菌活性。本研究结果将为进一步研究木霉几丁质酶的应用提供了基础。
译  名：
Cloning,prokaryotic expression and activity assay of chitinase(Tvchi) from Trichoderma viride
作  者：
YANG Li-rong1,SUN Hu1,LEI Zhen-sheng2,ZHAO Xian-lin2,QUAN Xin1,XUE Bao-guo1 (1Plant Protection Institute,Henan Academy of Agricultural Science,Henan Key Laboratory for Control of Crop Diseases and Insect Pests,IPM Key Laboratory in Southern Part of North China for Ministry of Agriculture,Zhengzhou 450002,China;2Research Centre for Wheat,Henan Academy of Agricultural Science,Zhengzhou 450002,China)
关键词：
Trichoderma viride;chitinase gene;gene cloning;prokaryotic expression
摘  要：
The cDNA of chitinase Tvchi gene was isolated from Trichoderma viride by using RT-PCR.Sequence analysis showed that the ORF of Tvchi was 1 293 bp in size and encoding 430 amino acid residues.The Blast showed that the Tvchi had high nucleotide sequences homology with other Trichoderma spp.Tvchi was further ligated with pET-28a vector and then transformed into Escherichia coli BL21(DE3).SDS-PAGE and Western-blot results revealed that Tvchi was expressed a 47 kD recombinant protein in E.coli.The imida-zole at the concentration of 100 mmol·L-1 could efficiently elute higher and purer purified protein.Affinity purification by Ni-NTA,high quality fusion protein was obtained.The optimum temperature and pH for reaction of the enzyme were 37℃ and 6.8,respectively.Analysis of antibacterial activity revealed that the expression products displayed better inhibiting ability on Fusarium graminearum Schw,Rhizotonia cerealis vander Hoeven and Gaeumannomyces graminis var.tritici Walker.The result applies a base for further research on chitinase Tvchi gene from T.viride.

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