费氏中华根瘤菌与耐盐有关的DNA片段的亚克隆和测序  

Subcloning and Sequencing of DNA Fragment Related to Salt Tolerance in Sinorhizobium fredii RT19

作　　者：卞学琳[1] 葛世超[1] 杨苏声[1] 

机构地区：[1]中国农业大学生物学院微生物学系,北京100094

出　　处：《Acta Genetica Sinica》2000年第10期925-931,共7页

基　　金：国家自然科学基金!(No.39270025);欧盟科研基金!(No.IC18CT960103)

摘　　要：将费氏中华根瘤菌（Ｓｉｎｏｒｈｉｚｏｂｉｕｍ　ｆｒｅｄｉｉ） ＲＴ１９与耐盐有关的２３ｋｂ ＤＮＡ片段用ＢａｍＨＩ酶切成大小不同的长度，分别与质粒 ｐＭＬ１２２连接，然后转化大肠杆菌（Ｅｓｃｈｅｒｉｃｈｉａｃｏｌｉ）Ｓ１７－１，筛选出３个转化子。以这些转化子为供体，ＲＴ１９的盐敏感突变株ＲＣ３－３为受体，分别进行二亲本杂交，筛选到接合子ＢＲ２，得到４．４ｋｂ与耐盐有关的ＤＮＡ片段。根据其物理图谱，酶切成６个ＤＮＡ片段，并分别连接到质粒ｐＵＣ１８进行测序。测序分析表明，该４．４ｋｂＤＮＡ片段含有ｆｉｘＯ、ｆｉｘＮ基因和３个开放阅读框（ＯＲＦ）。A 23kb DNA fragment related to salt tolerance was obtained from the gene library of S fredii strain RT19. In this study, BamHI was selected to digest 23kb DNA fragment into different length of DNA fragments. The resulting fragments were ligated with plasmid pML122, then the recombinant plasmids were transformed to competent cells of E. coli S17-1 on selective medium and three transformants TR were obtained. Two-parental mating experiments were carried out with these transformants as donor and salt sensitive S. fredii strain RC3-3 as recipient and the transconjugant BR2 was selected on FY plates containing gentamycin and 0.4mol/L NaC1. Thus, a 4.4kb DNA fragment related to salt tolerance was obtained. Based on its physical map, six restriction fragments were subcloned into plasmid pUC18 for DNA sequencing. Subsequently, sequencing and analysis of 4.4kb DNA fragment showed that fixO genes and three ORFs were obtained.

关 键 词：费氏中华根瘤菌 耐盐 亚克隆 测序 豆科植物 

分 类 号：Q933[生物学—微生物学]