绿色木霉内切葡聚糖酶基因Ⅰ的克隆表达

Cloning and heterogorous expression of EGI gene from Trichoderma viride

为深入研究绿色木霉(Trichoderma viride)内切葡聚糖酶(EGⅠ)的特性与功能,利用Northern blot方法分析不同碳源条件下绿色木霉的egⅠ表达,采用RT-PCR方法从绿色木霉T4中克隆egⅠ基因的cD-NA序列,测序与生物信息学分析,并构建诱导型表达载体pYES2-egⅠ,转化酿酒酵母(Saccharomycescerevisiae)INVSc1和H158中表达.结果表明:绿色木霉在含有纤维素的液体培养基中生长,egⅠ高效表达,在秸秆中表达量最高.纤维二糖中表达量相对较低,在葡萄糖、果糖中没有表达.egⅠ基因编码框长度1 377bp,编码459个氨基酸.含有信号肽,为分泌蛋白,属于糖苷水解酶家族7,具有纤维素酶结合域(CBD)和催化域(CD).INVSc1转化子发酵培养48 h达到转录高峰期,60 h达到酶活高峰期,酶活为0.081 6 U/mL,酶活比H158转化子提高32.5%.

To investigate the property and function of endoglucanse（EGⅠ）,the transcriptional expression of egⅠ of Trichoderma viride T4 was analyzed by Northern blot during its growth and development in medium with different carbon source.The full length cDNA sequence of egⅠwas obtained by RT-PCR method and the recombinant vector pYES2-egⅠ was constructed.The vector was transformed into Saccharomyces cerevisiae INVSc1 and H158.The results indicated that the egI was expressed strongly when T.viride T4 was grown in liquid medium containing cellulose as carbon source.The expression of egI was the highest in corn stalk medium,and cellobiose also induced the expression of egI,but no signal was detected in glucose or fructose medium.The ORF of egI was 1 377 bp encoding a protein of 459 amino acids and belonged to glycosyl hydrolase family 7 having cellulose binding-domain and catalytic-domain.egI was a kind of secreted protein because there was signal peptide in the amino acid sequence.The egI transcription of transformants reached highest at 48 hours and a peak recombinant enzyme activity was 0.081 6 U/mL at 60 hours,the recombinant enzyme of EG I of INVSc1 improved 32.5% than that of H158.