| Leguminous plants interacting with rhizobia establish a symbiotic relationship and form a highly specialized organ-nodule,which is the residence of the symbotic fixation rhizobia.The formation and development of nodules are the result of the interaction between rhizobia and host plants.In leguminous plants,there are two different forms of nodules:determinate nodules and indeterminate nodules.The meristem of the determinate nodules has a short activity cycle,and the developmental stages of the nodule tissues are the same;but the activity of the meristem of the indeterminate nodules is sustained,and the rhizobial bacteria continue to infect the meristem,resulting in a cell division of the nodule,infected areas,fixed nitrogen areas,and senescent areas,each area is at a different stage of development.The nodules of the model legume Medicago truncatula belong to indeterminate nodules.The symbiotic partner rhizobia begins to differentiate into bacteria in the infection zone and the nitrogen fixation zone and perform nitrogen fixation.In order to reveal the mechanism of infection,survival,colonization,and nitrogen fixation of rhizobia in root nodules,the study was conducted on the isolation and identification of symbiotic nitrogen-fixing gene mutants using Sinorhizobium meliloti 2011?Sm2011?as material.The main findings are as follows:1.Constructed a mutant library of Sm2011 strain:using three parental hybridization techniques,using Sm2011 as a recipient bacterium,E.coli S17-1/pMH1701 as a donor,and E.coli/pRK2073 as a helper.The results showed that the transposition frequency of Tn5-sacB was 1.84×10-6.After screening by Kan resistance,sucrose sensitivity and Tc resistance,about 8000 Tn5-sacB insertion mutants were isolated.Twenty-three single colonies were randomly selected and Tn5-sacB was confirmed by PCR in the rhizobial genome.2.Screening mutants of rhizobia-inhibiting immune-associated genes of root nodules in Medicago:Inhibition of the nodule immune gene mutant?nad1?results in the inability to form nitrogen-fixing nodules?white?when wild-type rhizobia are inoculated.Using the nodule phenotype of the mutant,the Tn5-sacB insertion mutant rhizobia were inoculated to screen pink nodules.The results of nodulation experiments showed that no pink nodules were found in about 1600 plants.This shows that using this method it is difficult to screen rhizobial mutants which could recover nitrogen-fixing activity in nad1 mutant nodules.3.Isolation and identification of symbiotic nitrogen fixation-related mutants of S.meliloti 2011:Using nested PCR detection technology,four specific target gene insertion sites and their mutants were identified and screened from approximately8000 strains of the Tn5-sacB insertion mutant,namely the nitrogenase gene nifH-75-9,the nodule related gene nodC-32-33,peptidoglycan related genes smc04024-76-79and smc01188-11-80,of which only the insertion site of smc04024 is located upstream of the start codon of the gene,and the other three mutants have Tn5 inserted in the gene reading frame.Through the nodulation experiment,the symbiosis and nitrogen fixation phenotypes of the mutants were examined.After the wild type Medicago seeds were inoculated with the nifH gene mutant for 28 days,no nitrogen-fixing nodules were formed?white?.Nodule formation was fully inhibited 10days after inoculation with the nodC gene mutant.This result is in excellent agreement with the previous research,confirming the integrity of the mutant pool.However,after 28 days of inoculation with smc01188 gene mutant,the obtained root nodule was pink,but the red area was greatly reduced compared with the wild type root nodule,and the back of the leaves was red-brown,and the plant showed a certain degree of nitrogen deficiency phenotype;After 28 days inoculation with smc04024gene mutant,the nodules and leaves of the plants were almost the same as those of the wild-type rhizobia inoculated,indicating that the insertion site of the gene did not destroy the normal expression of the gene.This provides resources and screening procedures for the identification of mutants of rhizobium,providing scientific data for the further study of the molecular mechanisms of symbiotic nitrogen fixation by Rhizobia. |
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