导入外源DNA片段的花生根瘤菌高效基因工程菌株的构建

CONSTRUCTION AND SCREENING HIGH EFFECTIVE STRAINS OF GROUNDNUT RHIZOBIA BY INTRODUCTING FOREING DNA FRAGMENTS

以柯斯质粒ｐＬＡＦＲ１为载体先构建快生型花生根瘤菌８５－７菌株总ＤＮＡ的基因文库。在协助质粒ｐＲＫ２０１３帮助下用此基因文库分别同菌株８５－７和另一株慢生型花生根瘤菌菌株Ｃ０２－５作三亲本杂交。转移接合子接种沙培条件下生长的花生，通过结瘤试验初筛选和复筛选，共筛选出３株工程菌ＨＮ１１、ＨＮ１２（以８５－７为受体）和ＨＮ１３（以Ｃ０２－５为受体）。其每植株根瘤的固氮酶活分别比出发菌株的提高３０２％（ＨＮ１１）、３５６％（ＨＮ１２）和２８４％（ＨＮ１３）；每植株干重分别比出发菌株的提高８５％（ＨＮ１１）、８３％（ＨＮ１２）和２８％（ＨＮ１３）。工程菌株的重组质粒酶切分析表明，除载体ｐＬＡＦＲ１外还分别含有２５ｋｂ（ｐＨＮ１１）、１４ｋｂ（ｐＨＮ１２和１８ｋｂ（ｐＨＮ１３）的外源片段。

We constructed a gene library of a fast-growth groundnut Rhizobium 85-7 in which 1.4×10￣(-4)Tcr clones were obtained and 57% of them proved to carry foreign DNA fragments. Then with the help of PRK2013 the gene library was seperately conjugated with the effective recipient strains 85-7 and a slow-growth groundnut Rhizobium co2-5. Mating efficiency was about 10￣(-4)-10￣(-5).The conjugates were inoculated into plants grown in sand culture.Three superior strains(named HN11,HN12,HN13)were obtained by comparing with the recipient strain 85-7(HN11,HN12)or co2-5 (HN13)on the basis of the acetylane reduction activity of nodules and plant dry weight.Their percentage increased were respectively 302 %(HN11),356%(HN12)and 284%(HN13)for the nitrogenase activity of nodule per plant and 85%(HN22),83%(HN12)and 28%(HN13)for the plant dry weight.The plant isolated from HN11.HN12 and HN13 was transformed into E.coli HB101.The plasmids isolated form HB101 transformants were disgested with EcoRI and about 25 kb,14 kb and 18 kb foreign DNA fragments were respectively in plasmids.pHN11,pHN12 and pHN13.