Construction Of Bradyrhizobium Diazoefficiens Mutants Library And Exploration Of Novel Functional Genes Related To Environmental Stresses

Leguminous plant-rhizobia symbiotic nitrogen fixation system is the most efficient system in biological nitrogen fixation,which is extremely important for global nitrogen ecological cycle and agriculture.Under natural or agricultural production conditions,the leguminous plant-rhizobium symbiotic nitrogen fixation system is continuously subjected to stress.Most studies have focused on nodule formation and nitrogen fixation mechanism of rhizobia in a controlled environment,as well as physiological and biochemical responses of leguminous plants or rhizobia to adverse conditions unilaterally.Whether and how rhizobium,as a closely symbiotic organism,helps plant fight against adverse environments remains unclear.It is of great significance to understand the biological role of B.d110 in the process of soybean response to drought stress injury and adaptive changes in order to improve agricultural yield and protect ecological environment.In this study,B.diazoefficiens USDA110 mutants library has been constructed by using Tn5 transposition mutagenesis.Several mutant strains probably involved in environmental stress have been screened,and their physiological,biochemical and genetic characteristics and symbiotic traits were preliminarily addressed.(1)The growth patterns of wild type B.d110 under three stress environments(pH,NaCl and drought)were first investigated.The growth of bacteria was apparently inhibited(critical growth)with the conditional treatment of pH4.0,0.4%NaCl and 12% PEG6000,respectively.Regardless of inoculation or not,soybean(Suinong no.14)showed significantly reduced biomass(dry weight of aboveground and root)under these treatments(environment stresses)by comparing with non-stress conditions.However,under stresses(pH and drought),inoculation with B.d110 significantly increased soybean biomass.In addition,photosynthetic rate(maximum photosynthetic rate and stomatal conductance)and water use capacity(maximum transpiration)of soybean leaves were significantly improved after inoculation with B.d110 under three stress conditions.The results showed that B.d110 is beneficial for soybean to resist the adverse environments.(2)In order to clarify the genetic background of B.d110 helping soybean to resist environmental stress,this study established a mutant library with a toal of 3842 strains by using Tn5 transposition mutation technology,and preliminarily identified the characteristics of the library.Nine mutant strains with distinct growth feature were screened under the conditional treatments as above.In addition,the amount and the precise location of Tn5 insertion site on the chromosomes of these mutant strains were determined by the Southern-blot and Inverse-PCR method.(3)Althought all these nine mutant strains showed somewhat different photypes from those of wild type B.d110,such as colony morphology,growth,motility,biofilm formation and others,only mutant strains 8-B6 and 8-H11 demonstrated obvious symbiotic defects with interaction with soybean.In these two mutant strains,gene coding for VirG two-component regulatory system and metal-bonding(Co)were inactivted by Tn5 insertion individully.The results of inoculation experiment under drought stress showed that the root nodules,physiology and nitrogen fixation ability of the plants under drought conditions were inferior to those of the soybean plants inoculated under normal conditions.It is inferred that these two genes may be related to stress function and further verification is needed.