费氏中华根瘤菌HN01的putA基因克隆  

Cloning of the putA gene from Sinorhizobium fredii HN01

作　　者：唐美琼[1,2] 申佩弘[1,3,4] 许兢[1,3,4] 蒋承建[1,3,4] 陈钢[1,3,4] 武波[3,4,5] 

机构地区：[1]广西大学生命科学与技术学院,南宁530005 [2]广西药用植物园,南宁530023 [3]微生物及植物遗传工程教育部重点实验室,南宁530005 [4]广西亚热带生物资源保护利用重点实验室,南宁530005 [5]广西民族大学化学与生态工程学院,南宁530007

出　　处：《应用与环境生物学报》2010年第5期697-700,共4页Chinese Journal of Applied and Environmental Biology

基　　金：国家自然科学基金项目(No.30570048)资助~~

摘　　要：从快生型大豆根瘤菌费氏中华根瘤菌HN01的基因组文库中克隆到一个putA(脯氨酸脱氢酶)基因,该基因与已报道的苜蓿中华根瘤菌1021中putA基因在核苷酸和氨基酸水平上分别有82%和86%相似性.利用自杀性质粒pK18mob构建含有putA基因部分片段的重组质粒,通过三亲本结合导入出发菌株HN01中,获得正向插入的极性突变体GXHNPA和反向插入非极性突变体GXHNPB.将putA基因完整的ORF连接到广宿主载体pLAFRJ上,获得用于互补的质粒pGXHN37,通过三亲接合,将pLAFRJ导入GXHNPA和GXHNPB获得互补菌株GXHNPHA和GXHNPHB.对出发菌株、突变菌株、互补菌株进一步的研究发现,以脯氨酸为唯一C、N源的MM培养基中培养时,突变体均不能生长,互补菌株和野生型HN01没有差异,而在完全培养基YMB中培养时,突变菌株生长不受影响.植株实验发现,突变体均能有效结瘤,但是固氮酶活与出发菌株相比有所下降,结瘤时间延迟1d,竞争结瘤能力下降,而互补菌株与野生型HN01没有明显的差异.A putA gene was isolated from the genomic library of Sinorhizobium fredii HN01 by molecular and biotechnological methods,and it had 82% and 86% identities with the sequence of the proline dehydrogenase from S.meliloti 1021 at nucleotide and amico acid level,respectivly.With the suicide plasmid pK18mob,the polar mutant GXHNPB and non-polar mutant GXHNPA inactivated in putA gene were constructed through homologous recombination.The putA gene was cloned into pLAFRJ vetor resulting in a complemention plasmid pGXHN37.The complement strains GXHNPHA and GXHNPHB were constructed by transforming pGXH37 into host strains GXHNPA and GXHNPB,respectively.The results of growth test showed that there was no difference among HN01,mutant and complement strains when were cultivated on completemedium YMB,but the mutants showed no growth in the MM medium containing proline as the sole carbon and nitrogen source.Plant tests revealed that mutants had lower nodulation efficiency ability and nitrogen-fixation,and nodulated 1 day later on soybean compared with the wild-strain HN01.

关 键 词：费氏中华根瘤菌HN01 putA基因 克隆 突变体 植株实验 结瘤 

分 类 号：S154.3[农业科学—土壤学]