谷氨酸棒状杆菌ATCC 14067阻遏蛋白ArgR与L-精氨酸合成途径相关基因的相互作用  

Interaction between Repressor Protein ArgR and Arginine Biosynthesis Pathway Related Genes in Corynebacterium glutamicum ATCC 14067

作　　者：敖婉钦 张豪 郑穗平[1] AO Wan-qin;ZHANG Hao;ZHENG Sui-ping(School of Bioscience and Bioengineering,South China University of Technology,Guangzhou 510640,China)

机构地区：[1]华南理工大学生物科学与工程学院,广东广州510640

出　　处：《现代食品科技》2019年第1期15-21,共7页Modern Food Science and Technology

基　　金：国家自然科学基金项目(31671840)

摘　　要：在细菌中阻遏蛋白ArgR通过与精氨酸合成途径相关基因中的保守序列结合,阻遏相关基因的转录。在谷氨酸棒状杆菌ATCC 14067中,精氨酸生物合成涉及的基因位于基因簇argCJBDFRGH和carAB中。构建重组菌株E.coli BL21(DE3)/pET22b-argR,诱导表达ATCC 14067来源的ArgR,成功得到目的蛋白。利用分子筛色谱柱对ArgR蛋白进行分析,表明ArgR蛋白相对分子质量在108 ku左右,在活性状态下ArgR蛋白为六聚体。合成生物素标记的谷氨酸棒状杆菌ATCC 14067来源的argC、argJ、argB、argF、argG、argH、carA基因片段,采用凝胶阻滞实验(EMSA)观察ArgR蛋白与其在体外的相互作用。结果表明,ArgR蛋白能够在体外与argC基因-26～+32 bp、argB基因-86～-29 bp、argG基因-161～-104 bp、carA基因-99～-42 bp结合,且与argB基因的结合程度最大,但不与argJ、argF、argH基因片段结合。本文首次将argC、argB、argG、carA基因中ArgR蛋白结合的靶标位点定位在60bp内,为更深入的研究谷氨酸棒状杆菌ATCC 14067中精氨酸合成的调控机制提供了理论依据。In bacteria,the repressor protein inhibits the transcription of arginine biosynthesis related genes through binding to conserved sequences in these genes.In Corynebacterium glutamicum ATCC 14067,the genes involved in arginine biosynthesis are located in argCJBDFRGH and carAB gene clusters.The recombinant E.coli BL21(DE3)/pET22b-argR was constructed to induce the expression of ArgR derived from ATCC 14067 and subsequently achieve the target protein.The analysis of the ArgR protein using molecular sieve columns showed that the relative molecular mass of ArgR protein was about 108 ku,and the ArgR protein was hexamer in the active state.The argC,argJ,argB,argF,argG,argH,carA gene fragments were obtained from biotin-labeled Corynebacterium glutamicum ATCC 14067,and their in vitro interactions with the ArgR protein were examined by electrophoretic mobility shift assays(EMSA).The results showed that the ArgR protein could bind to argC gene at positions-26^+32 bp,argB gene at positions-86^-29 bp,argG gene at positions-161^-104 bp,and carA gene at positions-99^-42 bp,and the degree of binding to the argB gene was the greatest whilst no binding between ArgR and the argJ,argF or argH gene fragment.For the first time,the target binding sites of ArgR protein in argC,argB,argG,and carA genes were located within 60 bp,which provided a theoretical basis for further study on the regulatory mechanism of arginine synthesis in Corynebacterium glutamicum ATCC 14067.

关 键 词：谷氨酸棒状杆菌 ArgR 分子筛色谱柱 凝胶阻滞实验 

分 类 号：Q78[生物学—分子生物学]