绿色木霉纤维素酶CBHII基因的分子克隆

MOLECULAR CLONING OF THE GENE CODING FOR CELLOBIOHYDRASE II FROM TRICHODERMA VIRIDE

本文以噬菌体lambda EMBL3 DNA为载体,通过克隆绿色木酶(Trichoderma viride)高分子量基因组DNA的部分酶解片段,并将重组分子进行体外包装后侵染Escherichia.coli K802,由此构建了绿色木霉基因文库。以李氏木霉(Trichoderma reesei)纤维素酶CBHII基因的末端片段为探针,用轮迥噬菌斑原位杂交从文库中筛选出CBHII基因的阳性克隆5个,随机取其中3个克隆用上述探针作斑点杂交,结果进一步证明克隆了全长或近全长的绿色木霉CBHII基因,用李氏木霉CBHI基因的末端片段探针作斑点杂交,结果提示CBHI与CBHII基因的末端序列之间无同源性存在。从斑点杂交的阳性克隆中提取DNA,酶切鉴定插入片段的长度,并克隆于质粒pUC19,Southern杂交结果证明获得了含绿色木霉CBHII基因的重组质粒pCBHII-14。

Gene library of Trichoderma viride was constructed in lambda EMBL3 DNA vector and screened by three rounds of plaque in situ hybridization with two probes representing 5'- or 3'-end sequences of cbh2 gene of Trichoderma reesei. Three of five positive clones were further tested by cross dot blotting with four probes respectively from 5'- or 3'-end sequences of cbh1 and cbh2 genes of Trichoderma reesei. It was confirmed that full-length cbh2 gene of Trichoderma viride had been cloned. However, homology in the end sequences had not been found between cbh1 and cbh2 genes of the Trichoderma. The length of inserts in vector EMBL3 DNA arms was identified by restriction enzyme analysis, and one of the inserts was then cloned into plasmid pUC19. It was convinced by southern blotting that the resultant recombinant plasmid (pCBH II-14) had contained cbh2 gene of T. viride.