导入四碳二羧酸转移酶基因对费氏中华根瘤菌共生固氮效率的影响

The Introduction of dctABD Genes into Sinorhizobium fredii and Its Effect on Symbiotic Nitrogen Fixation Efficiency

将来自苜蓿中华根瘤菌 (Sinorhizobiummeliloti)的四碳二羧酸转移酶基因(dctABD)经 pIJ2 92 5克隆到广宿主、稳定性质粒 pTR10 2上 ,获得诱导型表达的重组质粒 pHN2 0 2。在此基础上再引入来自 pDB30所含的发光酶基因 (luxAB)作分子标记 ,以 pTR10 2为基础构建成带有dctABD和luxAB的重组质粒 pHN2 0 5。经三亲本接合转移 ,将重组质粒 pHN2 0 5导入费氏中华根瘤菌 (Sinorhizobium fredii)HNO1,GR3和YC4。与出发菌相比较的盆栽试验结果表明 :HN0 1(pHN2 0 5)和GR3(pHN2 0 5)分别在宁镇一号和川早一号大豆上能显著提高植株地上部分干重 (生物量 )和总氮量 ,YC4 (pHN2 0 5)在黑龙 33大豆上能同时显著提高植株地上部分干重 (生物量 ) ,总氮量和根瘤鲜重。本研究结果表明 :导入dctABD基因对共生固氮效率的增效性与受体根瘤菌和大豆品种等因素有关。以luxAB为报告基因进行的转移接合子培养条件下分离单菌落和共生条件下形成根瘤的发光活性检测结果表明 :pHN2 0 5可在供试费氏中华根瘤菌中稳定遗传。

The 5 75kb DNA fragment containing C4 dicarboxylic acid transport ( dctABD ) genes isolated from Sinorhizobium meliloti was subcloned into the stable broad host plasmid pTR102 through pIJ2925, A inducible expression recombinant plasmid pHN202 was obtained The reporter gene luxAB from plasmid pDB30 was also inserted into pHN202 and the recombinant plasmid pHN205 was constructed and then introduced into Sinorhizobium fredii HN01, YC4 and GR3 by tri parental mating Plant pot experiments were carried out for the recipient strains and their transconjugants It was confirmed that recombinant strains HN01(pHN205) and GR3(pHN205) could significantly increase the shoot dry weight(biomass) and total nitrogen content of the plant with soybean varieties of Ningzhen N0 1 and Chuanzao No 1 respectively Transconjugant YC4 (pHN205) could also significantly increase the shoot dry weight(biomass), total nitrogen content of the plant and root nodule fresh weight with soybean variety Heilong 33 The results suggested that the effect of dctABD genes on the symbiotic nitrogen fixation efficiency was relevant to many factors such as soybean varieties, soil fertility and recipient rhizobia The results of colony and nodule lighting detection indicated that the recombinant plasmid pHN205 was stable in S fredii tested under both free living and symbiotic condition