Impact Of Sinorhizobium Fredii With Modified Expression Of PurL Gene On Symbiotic Abilities And Its Application In Biological Containment

The purine synthesis pathway plays an important role in the Rhizobium-plant interaction since most purine auxotrophs of rhizobia species are unable to form effective nodules on their host plants. Previous reports showed that a transposon induced PurL mutant of Sinorhizobium fredii induced pseudonodules on Glycine max, addition of 5-aminoimidazoie-4-carboxamide (AICA)-riboside or adenine and VBl to the plant could enabled the mutant to form infected nodules but no nitrogen fixation. To gain a better understanding of the impact of purL gene on the symbiosis formation, we studied the relationship between the purL gene and effective symbiosis on soybean by different transcriptional modification of S.fredii purL gene. The engineered genetic circuit of symbiotic-expressing purL was also applied in the biological containment study of rhizobia inoculum. In addition, other interested pleiotropic effects were studied with these PurL" mutants..Two different PurL" mutant, P825 and P618, were constructed from the wild type S. fredii strain HH103 by gene replacement. The strain P825 was the replacement mutant whose Xhol-Notl fragment in purL gene was replaced by luxAB, and the strain P618 was a 1.98kb deletion mutant in purL open reading frame. Both mutant required the supplement of adenine and VB1 to grow in minimal medium, and induced defective pseudonodules on soybean. Expressing vector pBBR-PG could restore the mutants to grow in minimal medium and the symbiotic abilities, which confirmed that they were the result of mutated purL gene. By the same method, four other PurL" mutants were constructed with S. fredii strain SMH12, HN01, WH3 and S11, to create the mutants SMH12-P1, HN01-P1, WH3-P1, S11-P1, SMH12-P2, HN01-P2, WH3-P2 and S11-P2, respectively. All these PurL" mutants were defective in symbiotic nitrogen fixation.Symbiotic expression vectors of purL gene using the promoter of fixK and fixR., pHN801K and pHN801R, could not restore the growth of P825 in minimal medium. In microaerobic stab inoculation in minimal medium, pHN801K and pHN801R could improve P825 a diffused growth like that of HH103, suggesting that purL could be expressed under the microaerobic conditions. However, neither two plasmids could restore the mutant to form infected nodules.New modification of purL expression containing a reduced expression plasmid(pHN811, 1/3 of the wild type level, constitutive) , symbiotic expression plasmids withpromoter of nifH, nifQ and fixN (pTPG-PnifH, pTPG-PnifQ and pTPG-PfixN), and aover-expression plasmid (pBBR-PG, 10 times of the wild type level, constitutive) wereconstructed. Plant results showed that the reduced expression of pHN811 and symbiotic...