黑曲霉基因文库的构建及葡萄糖淀粉酶基因的克隆(英文)

Construction of a Genomic Library and Cloning of Glucoamylase Gene from Aspergillus niger

用噬菌体λEMBL3 DNA为载体成功地构建了黑曲霉3758的完整的基因文库。供体黑曲霉染色体DNA经EcoRI部份酶切和蔗糖密度梯度离心,回收15—20kb片段,以一定的比例与经EcoRI处理的载体λEMBL3 DNA连接,连接产物用大肠杆菌SMR10单菌系统提取的包装蛋白进行体外包装,包装物感染宿主大肠杆菌Q359,获得2.5×10~5重组噬菌体/μgDNA的包装效率,比Clarke-Carbon公式对构建黑曲霉染色体基因文库要求的重组克隆数高10倍。以编码葡萄糖淀粉酶第329—481位氨基酸的DNA作为探针,用原位噬菌斑杂交的方法,从10~5个重组噬菌斑中筛选出2个杂交阳性噬菌斑。复筛以后,用快速法提取DNA,经EcoRI和EcoR V双酶切以后,走电泳,用Southern印迹法将DNA转移至硝酸纤维素膜上,与^(32)p-标记的探针杂交,证实葡萄淀粉酶基因位于2.5kb的EcoRI、EcoR V片段上,该片段已亚克隆至ρBR322。

A complete genomic library of Aspergillus niger was successfully con-structed using lambda EMBL3 DNA as vector. The donor A. niger DNA waspartially cleaved with EcoRI, 15-20kb fragments were recovered from sucrosegradient and ligated to the vector lambda EMBL3 DNA with T4-DNA ligase at aproper ratio and the ligated mixture was packaged in vitro with the packagingextract prepared from E. coli SMR10 single strain system. The packaged phageswere titered using E. coli 359 as host and 2. 5×10~5 recombinant phages/ug DNAwas obtained, which is 10x more than the number of recombinants required foran entire A. niger genome according to Clarke and Carbon. The glucoamylase genewas screened by in situ hybridization. The recombinant plaques were transferredto nitrocellulose filter and hybridize to ^(32)p labelled DNA probe, 2 positive pla-ques were obtained from 10~5 recombinant phages. After rescreening, DNA wasextracted from the positive phage for further analysis. The glucoamylase genewas localized on a 2. 5kb EcoRI-EcoRV fragment by Southern blotting. The 2. 5kbfragment was subcloned in RcoRI site of-EcoRV pBR322.