Screening And Gene Mining Of Nitrogen Fixation Deficient Mutants In Medicago Truncatula

Legumes can form a sustainable way of acquiring nitrogen resources with rhizobia in symbiotic nitrogen fixation.The success of the symbiotic nitrogen fixation system is attributed to the growth of new nodules in the roots of the host plant.Inside the nodules,the plant uses the nutrients synthesized by itself in exchange for the ammonia fixed by the rhizobia,and the two form a mutually beneficial symbiotic relationship.Many genes are involved in the process of root nodulation.It is particularly important to mine key genes in these processes and explore their functions.In this study,six Medicago truncatula retrotransposon insertion(Tnt1 insertion)mutants were analyzed through forward genetic methods,screening of mutants,establishment of genetic populations,whole genome sequencing,identification of candidate genes,phenotype analysis of mutants,genetic complementation of candidate genes,etc.We hope to discover new genes involved in the symbiotic process of nitrogen fixation and provide new ideas for further analysis of the symbiotic nitrogen fixation system.The research findings are as follows:1.The discovery of four nitrogen-fixing-deficient mutants and the establishment of their genetic populations.Phenotypic screening of six candidate mutants inoculated with rhizobia showed that three mutants formed white round nodules,and one mutant formed white columnar nodules.The reported genes were analyzed by PCR method,and no known genes of 4 mutants were found.The mutants were then backcrossed with wild-type R108 to establish genetic populations.2.Whole-genome sequencing analysis of three candidate mutants.According to the results of whole-genome sequencing of NF17605,NF17580,and NF9125,the sites with Tnt1 homozygous insertion were used as markers,and then the mutant phenotype individual plant in the F2 generation of the backcross populations was used to verify whether these markers were cosegregate with the phenotype.We found one marker that completely cosegregates with the phenotype of NF17605;three markers that co-segregate with the phenotype of NF17580 to a higher degree,and the three markers are close to each other;the marker that co-segregates with the phenotype of NF9125 has not yet been determined.3.The candidate gene of NF17605 is BID5(bacteroid with impaired differentiation 5).We named this mutant bid5,through bioinformatics analysis of the candidate gene,it was found that the gene encodes a WRKY transcription factor and is highly expressed in nodules.The phenotype of the mutants was further analyzed by inoculating rhizobia with different reporter genes,and it was found that the rhizobia in the nodules were alive,but they could not differentiate normally and had no nitrogen-fixing ability.4.The genetic complementation vector and spatiotemporal expression vector of bid5 were successfully constructed.BID5 can restore bid5 mutants to the wild-type phenotype.