哈茨木霉几丁质酶cDNA基因的克隆及在酿酒酵母中的表达

Cloning and expression in yeasts of the class V chitinase cDNA gene from Trichoderma harzianum

为研究哈茨木霉 (Trichodermaharzianum)的生物防治机制并获得与生物防治相关基因 ,通过构建哈茨木霉菌丝生长期的cDNA文库及对部分表达序列标签序列的测定与生物信息学分析 ,成功获得了哈茨木霉几丁质酶v(ChiV)基因的全长cDNA序列。该基因的编码框长度为 1194bp ,编码 397个氨基酸 ,理论分子量为 4 4kD。将该基因构建到酿酒酵母诱导型表达载体pYES2上 ,转化到酿酒酵母H15 8菌株中 ,通过Northern杂交检验后 ,确定该基因在酿酒酵母转录水平上表达。在 β_半乳糖诱导下 ,转化子在培养 6 0h时产生的酶活活性最高 ,几丁质酶V最适活性温度为 37℃ ,在pH 6和pH 8时活性较高。

To explore the integrated bio-control mechanism of Trichoderma harzianum and acquire some bio-control associated novel genes, a Trichoderma harzianum mycelium cDNA library has been constructed and thereby randomly selected clones were sequenced and analyzed by bioinformatics analysis. Full-length cDNA, encoding class v chitinase(ChiV), successfully cloned. The results indicated that the ORF of ChiV was 1194bp, encoding 397 aa, deduced molecular weight 44kD. The gene was ligated to the vector of pYES2 and thereby transformed to the yeast of H158 species. The enzyme activity reached the peak expression after cultured for 60h induced by beta- galactose. The optimal temperature of ChiV is 37℃ and the optimal pH is 6 and 8.