在大肠杆菌中直接克隆白腐丝状真菌-黄孢平革菌(Phanerochaete chrysosporium)基因启动子

DIRECT CLONING OF GENE PROMOTERS FROM A WHITE ROT FILAMENTOUS FUNGUS PHANEROCHAETE CHRYSOSPORIUM IN ESCHERICHIA COLI

利用启动子探钍型载体ｐＳＵＰＶ４ 直接在大肠杆菌细胞中克隆到多个来自白腐丝状真菌－黄孢平革菌（Ｐｈａｎｅｒｏｃｈａｅｔｅ ｃｈｒｙｓｏｓｐｏｒｉｕｍ ）的基因启动子片段．这些基因启动子片段能在大肠杆菌细胞中启动重组卡那霉素抗性基因（ｒｋａｎｒ）的表达，不同片段赋予宿主细胞以不同的卡那霉素抗性水平，最高的可达１０００μｇ／ｍ Ｌ，最低的则为５０μｇ／ｍ Ｌ．琼脂糖凝胶电泳显示这些重组质粒ＤＮＡ 中均有不同大小的插入片段，其范围是０．５～６．０ｋｂ．选取一个插入３．９ｋｂ 的重组质粒ｐＰＣ３３ 所作的Ｓｏｕｔｈｅｒｎ 杂交结果表明，插入片段以单拷贝形式存在于Ｐ．ｃｈｒｙｓｏｓｐｏｒｉｕｍ 的基因组中．当此片段５′－端被除去１．８ｋｂ 后，余下的２．１ｋｂ 片段可使融合的Ｋａｎｒ 基因的表达效率提高６０％ ．

A lot of gene promoter fragments have been cloned directly in Escherichia coli from a white rot filamentous fungus Phanerochaete chrysosporium by using a gene promoter probe vector pSUPV4.These fragments could promote the expression of recombinant kanamycin resistant gene (rkan r) in E.coli with different resistant levels.The highest kan r level was 1000μg/mL whereas the lowest one 50μg/mL.Result from agarose gel electrophoresis showed that all recombinant plasmids contained inserted fragments with the sizes between 0.5～6.0kb.An inserted fragment of 3.9kb in the recombinant plasmid pPC33 was chosen for Southern hybridization and the result proved that this fragment existed in single copy in the genome of P.chrysosporium. The expression efficiency of the modified rkan r gene increased 60% higher than that of original rkan r gene after the 1.8kb 5′ terminal segment of PC33 fragment was deleted out.