费氏中华根瘤菌lrp基因的克隆、突变与功能  

Mutation and Function of lrp Gene Cloned from Sinorhizobium fredii HN01

作　　者：陈钢[1,2,3,4] 唐美琼[1,2,3] 孙云[1,2,3] 许兢[1,2,3] 武波[1,2,3] 

机构地区：[1]微生物及植物遗传工程教育部重点实验室 [2]广西大学生命科学与技术学院 [3]广西亚热带生物资源保护利用重点实验室,南宁530005 [4]桂林师范高等专科学校,广西桂林541002

出　　处：《应用与环境生物学报》2008年第5期658-662,共5页Chinese Journal of Applied and Environmental Biology

基　　金：国家"973"计划(001CB108901);国家自然科学基金资助项目(No.30570048)~~

摘　　要：通过分子克隆技术,从费氏中华根瘤菌(Sinorhizobium fredii)HN01基因组文库中克隆到一个lrp基因.该基因与已报道的苜蓿中华根瘤菌(Sinorhizobium meliloti)1021的lrp基因在核苷酸水平上有89%相似性,在氨基酸水平上有99%相似性.利用自杀质粒pK18mob构建含有lrp基因部分片段的重组质粒,通过三亲本结合后导入原始菌株HN01中,经过同源单交换,获得发生正向插入突变的突变株GXHNLTA和反向插入突变的突变株GXHNLTB.将lrp基因ORF连接到载体pLAFR3上,获得用于互补的质粒pGXHNL100,将该质粒通过三亲本接合导入突变株中,获得互补株GXHNWA、GXHNWB.对野生型菌株、突变株、互补株进一步研究发现,在以脯氨酸、亮氨酸、丝氨酸等氨基酸为唯一碳、氮源的MM培养基中培养时,突变体GXHNLTA、GXHNLTB的生长均滞后于出发菌株HN01.植株试验表明,突变菌株GXHNLTA、GXHNLB比野生菌株HN01开始结瘤的时间提前1d,在结瘤效率、单株瘤数、瘤重、固氮酶活性方面并无显著差别.A lrp gene was cloned from a genomic DNA bank of Sinorhizobiumfredii HN01 through molecular biotechnology. Bioinformatics analysis showed that the lrp gene shared 89% homology at nucleotide level and 99% homology at amino acid level with the reported lrp gene orS. meliloti 1021. With the suicide plasmid pK18mob, the polar mutant GXHNLTB and nonpolar mutant GXHNLTA inactivated in lrp gene were constructed through homologous recombination. The integrate Irp gene was cloned into pLAFR3 vector resulting in a complementation plasmid pGXHNL100. The complementation strains GXHNWA and GXHNWB were constructed by transforming pGXHNL100 into host strains GXHNLTA and GXHNLTB, respectively. Both the non-polar and polar mutants of HN01 with lrp gene disrupted showed a little slower growth with respect to their parent type in the MM medium containing amino acids such as proline, leucine or serine as sole carbon and nitrogen sources. Plant tests revealed that mutants GXHNLTA and GXHNLTB showed one day ahead of the wild type in initial nodulation time and no obvious effects on other results such as nodulation efficiency and nitrogen-fixation.

关 键 词：SINORHIZOBIUM FREDII HN01 LRP基因 pK18mob 突变 植株试验 

分 类 号：Q93[生物学—微生物学]