在鱼腥藻7120中建立反义glnA系统  

The Establishment of Antisense GlnA System in Cyanobacterium Anabaena sp. PCC7120

作　　者：秦京东[1] 施定基[1] 徐旭东[2] 张金栋[3] 郭平仲[3] 汤佩松[1] 

机构地区：[1]中国科学院植物研究所 [2]美国密执安州立大学国家植物学实验室 [3]首都师范大学生物系,北京100037

出　　处：《植物生理学报（0257-4829)》1998年第3期225-232,共8页Acta Phytophysiologica Sinica

基　　金：国家自然科学基金

摘　　要：应用反义技术对鱼腥藻7120切的内源glnA基因的表达进行调控，首次获得了人工反义系统的蓝藻品系。先从编码谷酰胺合成酶（GS）的基因glnA中取得部分结构基因片段，与表达质粒载体pRL-439及穿梭质粒载体pDC－8相连接。通过酶切鉴定筛选出反向克隆的穿梭表达质粒pDC－AM，然后应用三亲接合转移法把它转入鱼腥藻对7120.通过新霉素筛选，酶谱鉴定，斑点杂交，质粒的交叉转化以及内源glnA基因表达的GS活性分析，GS相关的胞外泌氨分析及所获藻株的形态学变化，证明已在鱼腥藻7120中建立了人工反义glnA基因的品系。The metabolism of an organism can be regulated at different levels, of which the gene regulation is the most fundamental. Anificial antisense RNA system is able to inhibit the expression of a target gene, thus, it is an effective method to controI the metabolism. However, up to now, no artificial antisense RNA system in algae and cyanobacteria has been established. Ih this work, we tried to use the antisense technique to regulate the expression of endogenic gln A gene of Anabaena sp. PCC7120,and got an stain in which antisense glnA system had been established. First, a fragment of glnA gene coding glutamine synthetase(GS) was cut down and linked with an expressional Plasmid pRL-439 and a shuttle plasmid pDC-8 to get recombinants, and we screened antisense shuttle expression Plasmid pDC-ATGS by endonuclease digesting. Then it was transferred into Anabaena sp. PCC7120 by triparent conjugation transfer. Neomysin screening, endonuclease digestion, dot blotting, plashed cross transformation , observation of cell morPhologic changes, and determination of activities of glutamine synthetase and NH excretion of the cells were used to confirm that the strain had an artificialantisense RNA system. These results may have potential values for the improve now of industrial NH production.

关 键 词：反义glnA系统 GS 鱼腥藻7120 泌氨 代谢调控 

分 类 号：Q78[生物学—分子生物学] Q949.22