摘要
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.
出处
《四川大学学报(自然科学版)》
CSCD
1995年第6期732-737,共6页
Journal of Sichuan University(Natural Science Edition)
关键词
基因表达
环状芽胞杆菌
DAN
Xb片段
Bacillus circulans. gene expression regulatory fragment
forward enhancementaction
reversed attenuation action
Kan ̄r gene