利用Tn5 gusA5对大豆慢生根瘤菌lrp基因的诱变  

MUTAGENESIS OF IRP GENE FROM Bradyrhizobium japonicum BY TN5 gusA5

作　　者：唐咸来[1] 武波[2] 柏学亮[2] 唐东阶[2] 吕安国[1] 唐纪良[2] 马庆生[2] 

机构地区：[1]中国科学院沈阳应用生态研究所,辽宁沈阳110015 [2]广西大学农业部农业分子遗传重点开放实验室,广西南宁530005

出　　处：《微生物学杂志》2000年第4期18-21,共4页Journal of Microbiology

基　　金：国家高技术"863"计划资助项目!(863-101-03-03-01)

摘　　要：通过对重组质粒ｐＧＸＮ３００中的 ２．３ｋｂ ＥｃｏＲＩ片段测序分析发现，其上有一完整的ｌｒｐ基因和部分 ｐｕｔＡ基因，与 Ｋｉｎｇ ＮＤ等［１］报道的 Ｂ．ｊａｐｏｎｉｃｕｍ的ｌｒｐ基因ＤＮＡ序列有 ８８％同源性。利用 Ｔｎ５ ｇｕｓＡ５定位 诱变方法，对质粒ｐＧＸＮ３００进行插入诱变，得到２．３ｋｂ　ＥｃｏＲＩ片段上有Ｔｎ５ｇｕｓＡ５插入位点的质粒ｐＧＸＮ３００－ Ｔ３８，将ｐＧＸＮ３００－Ｔ３８转移到大豆馒生根瘤苗（Ｂ．ｊａｐｏｎｉｃｕｍ）ＧＸ２０１中，得到的ＧＸ２０１转移接合子与不相容 质粒ｐＰＨ１ＪＩ发生同源双交换。通过抗性及ｇｕｓＡ活性检测，筛选到一ｌｒｐ基因突变株。Ｓｏｕｔｈｅｍ杂交分析证 明这突变株的 Ｔｎ５ ｇｕｓＡ５插入确实是同源交换而不是转座产生，表明 Ｔｎ５ ｇｕｓＡ５ 诱变可以应用于大豆慢生根 瘤菌中的突变林筛选。kb of EcoRI fragment of recombinant plasmid pGXN300 was found to have a whole Irp'P gene and part of pu- tA gene after sequencing analysis. The Irp gene shares 88% homology Of DNA apuence with IrP genes of B. jatonicum reported by bang ND. The plasAnd was then mutagenital using Tn5 gusA5 orientation method and insertion mutagenesis. In addition, plasmid pGXN300-T38 with Tn5augA5 insertion in 2.3kb foreign DNA was constructed. PlasAnd pGXN300-T38 was then transferred to B. japonicum GX201. Homogenous exchange occurred in the obtained-transferred conjugated GX201 with incompatible plasmid pPH1JI. A Irp mutagenic strain was screened by resistant and gusA activity testing. Further analysis by Anthem hybridization has proven that the Tn5 gusA5 insertion in the mutagenic strain was a double homogenous exchange but not produced by transopition. These suggested that Tn5 gusA5 mutagenesis could be used in the screening of mutated strain of B. japonicum.

关 键 词：Tn5grsA5诱变 LRP基因 大豆慢生根瘤菌 

分 类 号：Q939.114[生物学—微生物学] Q78