Construction Of Highly Effective Recombinant Soybeam Rhizobium Strains And Study Of Their Micro-Ecology On Rhizosphere Colonization

5.75kb DNA fragment containing C4-dicarboxylic acid transport (dctABD) genes 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 also a recombinant plasmid pHN207 containing dctABD, parCBA/DE genes from pTR102 and reporter genes luxAB was constructed by using pLAFR3 as the vector. A recombinant plasmid pHN306 containing nifA of Klebsiella pneumonias and luxAB was also constructed by using pTR102 as the vector. And finally a recombinant plasmid pHN307 containing dctABD, nifA and luxAB genes was constructed.The recombinant plasmid pHN205, pHN207, pHN306 and pHN307 were then introduced into Sinorhizobium fredH-HNOl, YC4 and GR3 and Bradyrhizobium japonicum TA11 and CB1809 by tri-or bi-parental mating. A series of recombinant rhizobium strains were screened. Plant pot experiments were carried out for the recipient strains and their transconjugants to compare their nodulation ability and symbiotic nitrogen fixation efficiency. The results showed: (1) 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 No.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 nodule fresh weight with soybean variety Heinong 33. (2)The introduction of dctABD genes could significantly improve the symbiotic nitrogen fixation efficiency of TA11 and CB1809 with soybean varieties of Heinong 33, Ningzhen No.l and Yudou No.l. Compared with the control, the shoot dry weight (biomass) and total nitrogen content of the plants tested were significantly increased. (3)Recombinant strains HN01(pHN306) and GR3(pHN306) could significantly increase the nodule number, nodule fresh weight, shoot dry weight (biomass) and total nitrogen content of the plant with soybean varieties of Yudou NO. 1 and Heinong 33 respectively. Transconjugant YC4(pHN306) could also significantly increase the nodule number, nodule fresh weight and total nitrogen content of the plant with soybean variety Yudou No.l. (4) The introduction of pHN307 into the soybean rhizobia did not result in the expected further significant improvement effects on the symbiotic nitrogen fixation ability for all the recipient strains. (5) The soybean yield inoculated with recombinant strains of S. fredii with extra copy of dctABD gene was significantly greater than that of inoculated with recipient strains or nitrate fertilizer.The results of plant pot experiment and field experiment indicated that: the extra copy introduced dctABD gene or nifA gene could significantly increase the nitrogen fixation efficiency and the nodulation ability of the recipient strains respectively, and the effects were relevant to many factors such as soybean varieties, soil fertility and recipient rhizobia.By means of the luminescence detection, it was confirmed that all the recombinant plasmids constructed by pTR102 in this work were stable under free-living and symbiotic conditions, which proved that parCBA/DE genes could increase the stability of pLAFR3 in the transconjugants.The survival dynamics of two representative recombinant rhizobia strains HN01DL and TA113QD was similar to each other in the sterilized and non-sterilized pot-soil microcosms. It was proved that the survival dynamics of rhizobia tested in the sterilized pot-soil microcosms wassignificantly different from that of the non-sterilized pot-soil microcosms.The study on the relationship between rhizospheric colonization dynamics and nodulation in the non-sterilized rhizobox-soil microcosms suggested that: the luminescencent nodules formed by HN01DL were mainly located in the place of 0-4cm root segment from the coated seed, and it corresponded to the relativ...