Construction Of A Tn5-sacB-inserted Mutant Library Of Mesorhizobium Loti MAFF303099 And Screening Of Symbiosis-Related Mutants

The symbiotic nitrogen fixation between rhizobia and legume can avoid the abuse of industrial nitrogen fertilizer,and is of great value in improving agricultural production.The current research on symbiotic nitrogen fixation is still focused on its specific molecular mechanisms.The nod factor?NF?synthesised by rhizobia is the key signal molecule for the initiation of symbiosis.In addition,many genes in rhizobium are involved in the symbiotic process,which can ensure the normal differentiation of rhizobia to fix nitrogen?N2?,including the nod genes,nif genes,fix genes and some lps genes.The symbiosis between Mesorhizobium loti MAFF303099 and the model legume plant Lotus japonicus is highly representative of the symbiotic nitrogen fixation system and has important research value to reveal the mechanism of symbiotic nitrogen fixation.In the Mesorhizobium,MAFF303099 is the first to complete the whole genome sequencing.In this research we have constructed a random mutant library of M.loti strain MAFF303099,and a total of about 10,000 independent mutants were obtained,using transposon Tn5-sacB mutagenesis technique.We attemped to construct and screen the mutants in MAFF303099 associated with symbiotic to reveal symbiosis mechanism better.The main results are as follows:1.The growth curve and sensitivity to Km of MAFF303099 were determined,and the absorbance values of OD600 for the MAFF303099 in the best growth state was 1.0;the screening concentration of Km was 50?g /mL.2.The plasmid pMH1701 containing Tn5-sacB transposon was transferred to the target strain MAFF303099 by tri-parental mating,and the transformation efficiency was 3.75×10^-5.About 10,000 mutants were picked up and cultured with 96-well plates constructing the random mutant library of MAFF303099.3.By the identification of PCR,antibiotic and sucrose screening,we confirmed that the transposon Tn5-sacB was inserted into the genome of MAFF303099.Through TAIL-PCR in 16 mutants,10 mutants were identified the insertion site in the gene coding box,confirming the feasibility of the mutant library.4.A technique was constructed which can identify mutant rapidly from the library.The mutant of the symbiotic nodule gene nodB was identified,named M-nodB-1 through screening the mutant library,and we also identified the specific insertion site of Tn5-sac B in the nodB gene.5.The symbiotic related phenotype of M-nod B-1 indicated that MAFF303099 mutant of nodB could not form nodules on Lotus japonicus roots comparing with the wild type;histochemical staining of Lotus japonicus NINpro-GUS roots indicated that M-nodB-1 could also induce the expression of LjNIN and form the infection threads.But the expression quantity of LjNIN and the number of infection thread?IT?decreased significantly in comparison with the wild type.