华癸中慢生根瘤菌7653R MCHK_8182基因的共生固氮功能

Symbiotic nitrogen fixation function of MCHK_8182 in Mesorhizobium huakuii 7653R

根据华癸中慢生根瘤菌7653R全局转录组分析结果,获得在共生固氮阶段显著上调表达的基因MCHK_8182;设计重叠延伸PCR引物,构建MCHK_8182双交换突变重组质粒载体;通过三亲本杂交,将突变载体导入7653R;在蔗糖压力培养下进行筛选,最终获得MCHK_8182双交换突变株。PCR及测序验证双交换突变株构建成功。植物盆栽试验结果显示,与7653R野生型接种的植株相比,MCHK_8182突变菌株接种的植株根瘤数减少,但固氮酶活未见明显差异。

Based on analyzing the transcriptome of Mesorhizobium huakuii 7653 R,MCHK_8182was identified to be up-regulated significantly during the stage of symbiotic nitrogen fixation.SOE-PCR primers were designed and an MCHK_8182replacement vector was constructed.Through three parental matting,the replacement vector was introduced into M.huakuii 7653 R.The transconjugants were screened under a pressure of sucrose culture.The MCHK_8182replacement mutant was finally obtained and its correctness was confirmed by PCR and sequencing.The result of the pot experiment showed that the phenotype of host plants induced by MCHK_8182 mut had a remarkably reduced number of root nodules compared with wild-type 7653 R,but nodules nitrogenase activity remained unchanged.