哈茨木霉UBc基因的克隆及表达

Cloning and expressing of UBc gene from Trichoderma harzianum

筛选哈茨木霉的cDNA文库,克隆到泛素结合酶基因的cDNA全长序列,并对其进行生物信息学分析,该序列的长度为951 bp,包含一个455 bp的完整开放阅读框,编码157个氨基酸,其理论分子量为17.64ku.在氨基酸水平上,具有较高的保守性.以克隆载体pBK5313为模板,设计含有XhoI和BamH I酶切位点的引物,成功地获得包含完整阅读框的目的基因;将目的基因连接到表达载体pET28上,构建出含有泛素结合酶基因的重组子pET28-UBc,转化到大肠杆菌BL21(DE3)菌株中,成功地获得了大量的大肠杆菌转化子.通过对大肠杆菌转化子菌落PCR检验、质粒双酶切鉴定及测序分析,证实了成功转化.通过IPTG诱导,优化了泛素结合酶原核表达的条件,最佳诱导时间为4 h,诱导剂浓度对蛋白表达量影响不大.本研究为进一步研究哈茨木霉的生物防治作用机制,诠释蛋白质代谢途径提供基本技术支持.

A full-length eDNA of Ubiquitin-conjugating enzymes gene （UBc） was cloned by screening the cDNA library constructed by Trichoderma harzianum mycelium. The sequence was analyzed by bioinformatics methodology. The sequencing result including 951 bp showed that the ORF of UBc gene was 455 bp; 157 amino acids were encoded with highly conserved domain and the deduced molecular weight was 17.64 ku. The UBc gene with complete ORF was cloned by PCR with designed primers including XhoⅠ and BamHI sites according to the sequence from pBK5313. A recombinant vector with UBc gene inserts was constructed and thereby transformed to E. coli. BL21 （ DE3 ）. The transformants with UBc genes were identified by PCR in vitro, enzyme digested and sequencing to achieve successful transformation. The optimal expressing condition of the transformants induced by IPTG was cultured for 4 h, and varied IPTG concentrations were identical to the protein secrete amount.