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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。
The chitinase gene Tvchi was amplified from Trichoderma viride ZBS-6 by RT-PCR. The sequence analysis showed that the open reading frame of Tvchi was 1 293 bp, encoding 430 amino acid residues. Blast analysis showed that the open reading frame Chitinase 18 family endochitinase has high homology. The gene was cloned into the prokaryotic expression vector pET-28a and transformed into E.coli BL21 (DE3). After induced by IPTG, SDS-PAGE and Western blot analysis showed that 47 kD fusion protein was successfully obtained. The fusion protein was affinity purified by Ni-NTA column to obtain a high-purity fusion protein Tvchi. The optimum temperature for Tvchi was 37 ℃ and the optimum pH value was 6.8. The expression products showed good antibacterial activity against wheat eclipse, scab and sheath blight pathogens. The results of this study will provide the basis for further research on the application of Trichoderma chitinase.