黄孢原毛平革菌基因启动子的分离与鉴定

Isolation and Characterization of Promoters from Phanerochaete chrysosporium

利用启动子探针型载体 pSUPV8直接在大肠杆菌 (Escherichiacoli)中分离黄孢原毛平革菌 (Phanerochaetechrysosporium)基因启动子片段 ,获得 6个潮霉素抗性 (Hygr)重组子。对重组子CH2、CH6进行序列分析 ,结果发现它们都存在真核生物基因启动子的保守序列 ;用原生质体转化法将其转化黄孢原毛平革菌 ,仅 pCH6获得了潮霉素抗性转化子 ;PCR和斑点杂交分析表明 ,pCH6已成功导入黄孢原毛平革菌 ,并启动潮霉素抗性基因的表达。

Promoter probe vector pSUPV8 was used to clone promoters from Phanerochaete chrysosporium directly in Escherichia coli . Six hygromycin B resistant recombinants were obtained and two inserted fragments of them were sequenced. The results indicated that they contain several sequences similar to eukaryotic cis regulatory elements. Only pCH6 could transform P.chrysosporium into hygromecin B resistance. PCR and dot hybridization analysis indicated that it was introduced into P.chrysosporium successfully and leaded to the HmB resistance. The results demonstrated that HmB resistant gene ( hph ) could be used as an ideal reporter gene for P.chrysosporium transformation.