哈茨木霉Cu-ZnSOD的克隆表达及活性鉴定

为研究哈茨木霉(Trichoderma harzianum)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超氧化物歧化酶(Cu-Zn SOD)基因的全长cDNA序列.该基因的编码框长度为465 bp,编码154个氨基酸,理论分子量为15.7 ku.将该基因构建到表达载体pET28上,转化到大肠杆菌BL21菌株中,通过诱导表达条件的优化后,经裂解菌体取上清进行酶活鉴定,确定在蛋白水平上得到了表达.蛋白质表达的最佳条件为:IPTG浓度为0.125 mmol/L,OD600为0.600,培养温度37℃,诱导时间为4 h.目的蛋白SOD的粗酶活为26.69 U/mL.该研究结果为进一步研究哈茨木霉的抗逆境胁迫分子生物学机制奠定基础.

Cloning, expression and biological activity identification of Cu-Zn SOD from Trichoderma harzianum

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 Cu-Zn SOD was successfully cloned.The results indicated that the ORF of Cu-Zn SOD was 465 bp,154 amino acids were encoded,and the deduced molecular weight was 15.7 ku.The gene was ligated to the vector of pET28 and thereby transformed to the E.coli BL21.After screening the correct recombinant E.coli pET28a-SOD/BL21 transformants,it was expressed by IPTG and the expressed products were analyzed with SDS-PAGE.The expression condition was optimized to obtain the maximum quantity of SOD.And then the protein activity was detected by SOD kit.The optimization condition for SOD expression was as follows: cultivating at 37 ℃ and initial induction at OD600 of 0.6 with IPTG of 0.125 mmol/L for 4 h.The protein activity was 26.69 U/mL.The SOD gene was expressed successfully and the recombinant protein was obtained.This study provides the foundation for studying the molecular mechanism of Trichoderma harzianum in future research.