【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。更多还原

【Abstract】 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.更多还原