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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 four-carbon dicarboxylic acid transferase gene (dctABD) from Sinorhizobium meliloti was cloned into broad host and stable plasmid pTR10 2 by pIJ2 92 5 to obtain the inducible recombinant plasmid pHN202. On the basis of this, the luxAB gene from pDB30 was introduced as a molecular marker and the recombinant plasmid pHN205 with dctABD and luxAB was constructed based on pTR10 2. After the transfer of the three parents, the recombinant plasmid pHN205 was introduced into Sinorhizobium fredii HNO1, GR3 and YC4. Compared with the starting bacteria, the pot experiment showed that HN0 1 (pHN2 0 5) and GR3 (pHN2 0 5) could significantly increase the shoot dry weight (biomass) of Ningzhao No.1 and Chuanzao No.1 soybean, And total nitrogen, YC4 (pHN205) could significantly increase aboveground dry weight (biomass), total nitrogen and nodule fresh weight in Heilong 33 soybean at the same time. The results of this study show that the synergistic effect of introduction of dctABD gene on symbiotic nitrogen fixation is related to factors such as receptor rhizobia and soybean varieties. The result of luminescent activity assay showed that pHN205 could be inherited stably in S. ruthenicus tested with luxAB as a reporter gene and isolated from single colonies and symbiosis.