粗毛栓菌(Trametes gallic)基因启动子的分离与鉴定

Isolation and Characterization of Promoters from Trametes gallic

粗毛栓菌 (Trametesgallic)总DNA经Sau3AI酶切后插入到启动子探针型载体pSUPV8的BamHI位点 ,在含氨苄青霉素与潮霉素双抗平板上筛选到 8个潮霉素抗性(Hygr)重组子 (命名为 pTP1～ 8) ,其插入片段大小为 0 .5～ 1.7kb ,抗性水平为 0 .15× 10 - 3 ～0 .35× 10 - 3 g/mL .对 pTP6进行酶切分析表明 ,插入片段中含有KpnI ,XbaI和SacI位点各一个 ,用限制酶SacI和XbaI去掉其 5’端 0 .7kb和 1.2kb片段后 ,其潮霉素抗性由 0 .2 5×10 - 3 g/mL均降为 0 .15× 10 - 3 g/mL .将TP5片段与粗毛栓菌总DNA进行Southern杂交 ,结果证明 ,TP5片段来源于粗毛栓菌总DNA .对片段TP5的 3’端进行序列分析 。

DNA fragments obtained from Sau3AI partially digested total DNA of Trametes gallic were cloned into BamHI site of pSUPV8,a promoter-probe vector.The recombined DNA was transformed into Escherichia coli cells and 8 hygromycin B-resistant recombinants were obtained(named pTP1～pTP8).The length of inserted fragments ranges from 0.5 to 1.7kb and the hygromycin B-resistant level ranges from 0.15×10 -3 to 0.35×10 -3g/mL. Restriction mapping of pTP6 indicated that there is restritcion enzyme site for KpnI,XbaI and SacI.After deleting the 0.7kb and the 1.2kb of 5'-terminal segment of pTP6,which has a hygromycin B-resistant level of 0.25×10 -3g/mL,the resulted subclones both have a lower hygromycin B-resistant level of 0.15×10 -3g/mL. Southern hybridization using TP5 as probe showed that TP5 can hybrid with the total DNA of T. gallic but not with that of E. coli.The 3'-terminal segment of TP5 fragment was sequenced, The result indicates that it contains several sequences similar to both prokaryotic and eukaryotic gene promoter sequences.