环状芽胞杆菌（Bacillus circulans）基因表达效率调节DNA片段的研究

STUDY ON AGENE EXPRESSION REGULATORY FRAGMENT FROM BACILLUS CIRCULANS

用大肠杆菌（Ｅｓｃｈｅｒｉｃｈｉａｃｏｌｉ）启动子探针型载体ｐＳＵＰＶ１从环状芽胞杆菌（Ｅａｃｉｌｌｕａｅｉｒｃｕｌａｎｓｃ－２）基因组中分离的启动子片段ＢＣ６能使无启动子的卡那霉素抗性基因恢复活性，并在大肠杆菌中获得高效表达（卡那霉素抗性高达１２００μｇ／ｍＬ）．当用限制酶ＸｂａⅠ去除其５′－端１．２ｋｂ片段（命名为Ｘｂ片段）后，３′－端片段仍能启动Ｋａｎ￣ｒ基因表达，其抗性水平降至４００μｇ／ｍＬ但当１．２ｋｂ片段反向插回原有位置时，其抗性水平降至６００μｇ／ｍＬ．将Ｘｂ片段正向插入另一重组质粒ｐＢＣ３中融合的Ｋａｎ￣ｒ基因启动子的上游时，卡那霉素抗性由２００μｇ／ｍＬ上升至１０００μｇ／ｍＬ．当Ｘｂ片段反向插入时，Ｋａｎ￣ｒ基因的表达却明显受到抑制，降为１００μｇ／ｍＬ．这说明Ｘｂ片段具有正向增强而反向衰减Ｋａｎ￣ｒ基因表达的能力．用ＢＣ６片段的两个亚克隆片段与ｃ－２菌株总ＤＮＡ分别作Ｓｏｕｔｈｅｒｎ杂交时，结果显示３′－端片段以单拷贝形式存在于基因组中，而Ｘｂ片段则以多拷贝形式散布其中．由此推断Ｘｂ片段并非一定伴随该基因启动子存在．

This paper describes the novel trait of a promoter-contained fragment BC6(2.4kb)isolated from Bacillus circulans with a promoter- probe vactor pSUPV1. This fragrnent could make apromoterless kanamycin-resistant gene express in Esheruichia coli with high efficiency up to1 200μg/mL. When its′-terminal 1.2kb segment(designated Xb fragment)was cut away withthe restriction enzyme XbaI,the rernained 3′-terminal segrnent could also initiate the expression ofthe promoterless Kan￣r gene,but whose expression efficiency reduced at least 3-fold(dowm to400μg/mL). When Xb fragment was forward inserted into the upstream of thehsed Kan￣r gene inanother recombinant plasmid pBC3,the expression level of the resulted Kan￣r gene was found toincrease 5-fold(from 20μg/mL to 1 00μg/mL).Howerer.when Xb fragment was inserted at thesame position of pBC3 with reverse orientation,it surprisingly showed that the expression efficiencyof the Kan￣r gene was obviously reduced(down to 100μg/mL).In the light of the above results aconclusion could be drawn that the Kan gene expression efficiency could be forward enhanced butreversely attenuated by the Xb fragment.When the Xb fragment and its downstream fragment wereused for Southern hybridiration with the total DNA of B.circulans c-2 completely digested withdifferent restriction enzymes,respectively.it was showed that the 3′-terminal fragment existed in thegenome with single copy,whereas Xb fragment did with multicopies.We could infer that Xbfragment does not necessarily accompany the promoter sequence. Not only does the Xb fragmentwell facillitate for us to reveal the regulatory mechanism of bacterial gene expression, but also couldbe utilized to construct expression vector with high efficiency.