绿色木霉LTR-2 β-1, 3-葡聚糖酶基因的克隆及其在 毕赤酵母中的表达

Cloning of β-1,3-glucanase gene from Trichoderma virid LTR-2 and its ex-pression in Pichia pastoris

摘要:

根据从GenBank中检索到的木霉菌β-1, 3-葡聚糖酶基因序列设计引物, 以高产β-1, 3-葡聚糖酶菌株——绿色木霉LTR-2的cDNA为模板, 采用PCR方法扩增得到内切β-1, 3-葡聚糖酶基因(glu)。将glu克隆至载体pMD18-T上, 进行了全序列测定。序列分析表明该基因由2289个核苷酸残基组成, 含有一个开放阅读框架, 可以编码762个氨基酸, 与报道基本相同。翻译后的氨基酸序列含有两个β-1, 3-葡聚糖酶的保守区RVVYIPPGTY和AASQNKVAYF。基因与已发表的木霉β-1, 3-葡聚糖酶基因有较高的同源性, 其中和哈茨木霉bgn3.1和绿木霉bgn13.1的同源性达到93%。序列已经提交GenBank, 登录号为EF176582。将glu基因插入到巴斯德毕赤酵母(Pichia pastoris)穿梭载体pPIC9K中, 获得重组质粒pGLU14, 经线性化后转化毕赤酵母菌株KM71。经大量平板筛选, 获得能有效分泌表达β-1, 3-葡聚糖酶的毕赤酵母工程菌株KGLU14, 菌落PCR扩增证实了glu基因已经整合到酵母基因组中。SDS电泳结果表明其β-1, 3-葡聚糖酶的分子量大约为80kDa, 和理论推测值大致相同。摇瓶发酵结果表明, 培养基中β-1, 3-葡聚糖酶的活力可达889U/mL。

关键词:β-1, 3-葡聚糖酶, 克隆, 序列分析, 分泌表达

Abstract:

Text We designed a pair of primers according to fungal glucanase genes obtained from GenBank and cloned a novel β-1,3-glucanase gene (glu) from Trichoderma virid LTR-2 cDNA by PCR. Then we linked the fragment with pMD18-T vector and sequenced it. The sequence analysis indicated that glu was composed of 2289 nucleotide residues. The fragment contained an Open Reading Frame coding 762 amino acids and was similar to previous reports. Translated amino acids sequence of glu contained two Conservative Districts of β-1,3-glucanase which were RVVYIPPGTY and AASQNKVAYF. By nucleotide blasting in NCBI glu showed high homology to three β-1,3-glucanase genes from Trichoderma sp., especially with T.harzianum bgn3.1 and Hypocrea virens bgn13.1, which the homology reached 93%. The sequence was submitted to GenBank and the Ac-cession Number is EF176582. Then we connected glu gene with the Pichia pastoris shuttle vector-pPIC9K. The recombinant plasmid named pGLU14 was transformed into methylotropic yeast P. pastoris KM71 after linearization. The recombinant strain KGLU14 expressing β-1,3-glucanase at high level was obtained through plate screening. The SDS-PAGE result indicated that molecular weight of the recombinant β-1,3-glucanase was about 80kDa and the activity of the recombinant enzyme could reach 889U/mL in liquid culture.

Key words:β-1, 3-glucanase; clone; sequence analysis; expression of secreted