| Rhizobia interact with most legumes to induce the formation of plant nodules where nitrogen gas is reduced into ammonium.It was found that one key step determing the specificty-of legumes rhizobial symbiosis is the response to specific flavonoid compound from legumes by rhizobial NodD protein,a regulator required for expression nod ABC gene for biosynthesis of nodulation factor.Nodulation factors are then recognized by plant receptor proteins initiate nodulation signal transduction in legumes leading to the formation of nodules.However,these results regarding NodD responding to specific falvonoids are indirectly inferred from a large number of genetic data,with the direct binding data lacking.This thesis main studied the interactions between NodD proteins and different flavonoids molecules.In this work,nodD1 and nodD2 genes were cloned from 6 rhizobia strains including soybean rhizobia,Medicago rhizobia and Lotus japonicus rhizobia.We selected four widely used flavonoids: i.e.,naringenin,genistein,daidzein,luteolin,and studied their interaction with different NodD proteins.Isothermal titration calorimetry(ITC)was used to determine the heat changes during the interaction between wild type and different truncated NodD proteins and four flavonoid molecules.Based on the change of heat,it was found that the C-terminus of NodD protein has the key residues for flavonoid binding.These nodD genes were introduced into the mutant Sinorhizobium fredii HH103 nodD2 to verify the biological function of the key site of NodD based on the nodulation phenotype in soybean.The results are included as follows:1.12 nodD genes were cloned fromin 6 strains(each strain contains nodD1 and nodD2 genes)and constructed into protein expression vector.Based on the amino acid sequence alignment and predicted of tertiary structure of NodD proteins,we designed four truncated amino acid sites at the C-terminus of nodD1 and nodD2 gene from S.fredii HH103,respectively.The four truncation sites are 128F-132 P,146V-152 P,192S-198 P and 246P-249 L.And the four truncated genes were constructed into protein expression vectors for protein expression and purification in prokaryotic system.Through screen of protein expression conditions,the NodD1 and NodD2 proteins in S.fredii HH103 with good expression levels were chosen for further study.2.The binding data of four truncated NodD2 proteins(C21-C24)and four flavonoids showed that: the truncated proteins(C21-C24)could not correctly recognize naringenin,genistein and luteolin,and the two combined with no obvious heat produce;The combination of truncated protein C23 and daidzein has obvious heat generation,that is,the deletion of 192S-198 P amino acid does not affect the recognition of daidzein by NodD2 protein,and the truncated protein(C21,C22,C24)cannot correctly recognize daidzein.The above data indicate that the truncated amino-terminal sequences128F-132 P,146V-152 P,192S-198 P and 246P-249 L may all be involved in the recognition of naringenin and genistein by NodD2 protein;128F-132 P,146V-152 P and246P-249 L was involved in the recognition of daidzein by NodD2 protein.Luteolin(unmatched flavonoid)is not specifically recognized by NodD1 and NodD2 proteins.3.In order to verify the biological functions of the above four key sites,we successfully knocked out the nodD2 gene in S.fredii HH103 through three-parent hybridization and two-parent hybridization experiments,and obtained an insertion mutant S.fredii HH103ΩnodD2.We complemented the four truncated nodD2 genes and the full-length nodD2 gene into S.fredii HH103ΩnodD2 strain respectively,soybean plants were inoculated and the phenotype were observed,in the phenotypes of the four truncated gene mutants of nodD2 gene,the number of nodules in the roots of soybean seedlings in the same batch was lower than that of the wild type.It is suggested that these sites play an important role in nitrogen fixation of nodules.The molecular mechanism of the interaction between NodD protein and flavonoid was verified in vitro by ITC and other technical means and some key sites were obtained based on the genetic analysis about them,so we can change the nodD sequence by gene knock-out and gene mutation,which provides a new way to expand the broad-spectrum of rhizobia. |
