Study On The Transfer Of Indigenous Plasmid In Rhizobia Between Genera And Diversity Of Rhizobia

In this work,Mesorhizobium huakuii 2020 contains three indigenous plasmids named p2020a,p2020b and p2020c,respectively.The other Mesorhizobium huakuii 7653R with two mega-plasmids,p7653Ra and p7653Rb,was stored in our lab.They were labeled by Tn5-mob-sacB insertion and the mutant strains labeled in each plasmid were obtained.Plasmid curing experiment was carried out by using the sacB positive selection method and plasmid cured derivatives,2020D29,2020D8 and 7653RD were obtained. 2020D29 was the mutant in which the first large plasmid p2020c was cured and 2020D8 was the mutant cured with the second plasmid p2020b.7653RD was the mutant cured with the symbiotic plasmid p7653Rb.Furthermore,the plasmids p2020a in 2020 and p7653Ra could hardly be eliminated which suggested that some house-keeping genes necessary for strain growth located on those plasmids.The results of plant nodulation tests demostrated that the mutant 2020D29 could significantly enhance the capacity of symbiotic nitrogen fixation.But the mutant 2020D8 lost the ability to nodulate Astragalus sinicus.Then the Sym plasmid pJB5JI of R.leguminosarum by.viciae T83K3 was transferred into M.huakuii 2020 and 7653R and their plasmid-cured derivatives 2020D29, 2020D8,and 7653RD.A set of transconjugants were obtained.And the transconjugants 2020-137,2020D8-8,7653R-197 and 7653RD35 in which the size of the exogenous plasmid was the same as pJB5JI.The transconjugants 2020-122,2020D29-13,2020D8-80, 7653RD5 and 7653R-15 in which the exogenous plasmid was smaller than pJB5JI.The results of pot plant tests showed that the ability of competition and symbiotic nitrogen fixation of transconjugants 2020-137 and 7653R-197 were significantly increased comparing with 2020 and 7653R.pJB5JI could not restore the ability of 2020D8 and 7653RD to nodulate their host plants which showed the function of pJB5JI could not restore the function of their original Sym plasmid.Of all the transconjugants 2020D8-8 and 7653RD35 could only form few white nodules on pea plants which showed that pJB5JI could express its function under the chromosomal background of Mesorhizobium huakuii.The other transconjugants failed to nodulate pea plants and the reasons might originated from the interference between double symbiotic plasmids.The stability of exogenous plasmid was checked in transconjugants under both free-living and symbiosis. pJB5JI was failed to be detected in some nodule isolates from Astragalus sinicus and pea. Km resistance gene could be amplified from all transconjugants and nodule isolates which suggested that pJB5JI might fully or partially integrated into the chromosome of recipients.Sinorhizobium fredii HN01 contains three large plasmids and the second large plasmid is its symbiotic plasmid.There are three derivatives HND29 cured with the symbiotic plasmid pSymHN01b and HND28 cured with pHN01c and HND100 cured with pSymHN01b and pHN01c,pJB5JI was transferred into HN01 and its mutants.A set of transconjugants were obtained.The transconjugants HN01-92,HND29-6,HND28-30 and HND100-123 in which the size of the exogenous plasmid was the same as pJB5JI. The transconjugants HN01-35,HND29-5 and HND28-43 in which the exogenous plasmid was smaller than pJB5JI.And the transconjugant HND28-66 which contained two exogenous plasmids.One was same as pJB5JI and the other was same as the smallest plasmid in T83K3.The results of pot plant tests showed that the symbiotic ability of transconjugants HN01-92 and HND28-30 were same as the original strains.pJB5JI could not restore the ability of HND29 and HND100 to nodulate their host plants which showed the function of pJB5JI could not restore the function of their original Sym plasmid.All the transconjugants failes to nodulate pea plants which showed that pJB5JI couldnot express its function under the chromosomal background of Sinorhizobium fredii.The stability of exogenous plasmid was checked in transconjugants under both free-living and symbiosis.the exogenous plasmid was very stable in the transconjugants of HN01-92, HN01-35,HND28-30,HND28-43 and HND28-66 through the symbiosis with soybean.Diversity of 42 isolates from effective nodules of Pisum sativum in different geographical regions of China were studied by using 16S rRNA gene RELP patterns,16S rRNA gene sequencing,16S-23S rRNA intergenic spacer(IGS) region RELP patterns and G-C rich random amplified polymorphic DNA(RAPD).The isolates were distributed in two groups on the basis of their 16S rRNA gene RFLP patterns and the 16S rRNA gene sequences of strains from 16S rRNA gene RFLP patterns.Groupâ… was very closely related(identities higher than 99.5%) to Rhizobium leguminosarum USDA 2370.Groupâ…¡consisting of WzP3 and WzP15 was closely to Rhizobium etli CFN42.The analysis of the 16S-23S IGS RELP patterns divided the isolates into 18 genotypes and four groups. Groupâ… was clustered with R.leguminosarum USDA2370.Groupâ…¡consisted of YcP2, YcP3 and CqP7.The strains of groupâ…¢distributed abroadly.Groupâ…£consisted of WzP3,WzP15 and R.etli CFN42.RAPD divided the isolates into nine clusters in which groupâ…£only consisted of YcP2.Strains of groupâ…¤andâ…¨were from Wenzhou and Xiantao,respectively.This assay demonstrated geographical effect on genetic diversity of pea rhizobia.