【摘要】 用绿色木霉核DNA部分酶解片段与噬菌体λEMBL3载体左右臂连接,并用高效价包装蛋白对重组分子进行体外包装,侵染宿主菌后,构建了含1.92×10₆个重组噬菌体的绿色木霉基因组文库。以李氏木霉纤维素酶CBH Ⅰ基因的5′、3′两个末端片段为探针,对所建立的基因组文库作轮回噬菌斑原位杂交,筛选到含绿色木霉CBH Ⅰ基因的阳性克隆7个,随机取其中三个克隆进一步用李氏木霉CBH Ⅰ、CBHⅡ基因的5′、3′四个末端片段探针作交叉斑点杂交,证明本实验确实克隆了很可能为全长的绿色木霉CBH Ⅰ基因,并提示CBH Ⅰ与CBHⅡ基因的末端序列之间不存在同源性。更多还原

【Abstract】 High-molecular-weight genomic DNA of Trichoderma viride were partially digested with MboI and conditions for recovering the suitable fragments were optimized. Bacteriophage lambda EMBL3 arms were prepared to clone foreign fragments. In vitro packaging was then performed using high-titer packaging proteins after the ligation of recovered fragments to vector arms. The resultant genomic library contained 1.92×10~6 recombinants.The amplified genomic library was screened by three rounds of successive plaque in situ hybridization with two probes representing 5′-or 3′-end sequences of cbh1 gene of Trichoderma reesei, which identified 7 positives corresponding to cbh1 gene of T. viride, Those positive clones were further tested through cross dot blotting by using four probes from 5′-or 3′-end sequences of cbh1 and cbh2 genes of T. reesei. It was confirmed that up-to-full-length cbh1 gene of T. viride had been cloned. However, homology in the end sequences had not been found between cbhl and cbh2 genes of the Trichoderma.更多还原