| Legume plants can form nitrogen-fixing symbioses with rhizobia in soil,which enables plants to grow on atmospheric nitrogen in nitrogen deficient environment.Biological nitrogen fixation occurs in this specialized organ,called nodule.Intracellular bacteroids compose an organelle called the symbiosome together with the host membrane and undergo terminal differentiation to form mature bacteroids in infected cells.For the past 20 years,research on nitrogen fixation has focused on the discovery of genes involved in the nitrogen fixation process by both forward-and reverse-genetics approaches.Compared with thousands of genes highly expressed in nodules in the whole genome expression database of legume,the current exploration is probably only the tip of the iceberg.There is still a huge space for the exploration of genes involved in the nitrogen fixation process.Based on the traditional forward-genetics approach,we begin our study with the mutant phenotypes,and then find the genes involved in nitrogen fixation symbiosis by map based cloning and analyze the biological functions of these genes.It can provid new directions for analysis of the signaling pathway of nitrogen fixation.The main findings is as follows:1.By screening nodule phenotype of 8 FNB mutants and excluding known genes,we find that two mutants of fn8013 and fn9133 is defective in nitrogen fixation and one mutant of fn7852 is defective in nodule formation.2.After inoculated with three different rhizobia in green zeolite,the aboveground growth rate of fn8013 is inhibited.fn8013 form small brown nodule when inoculated with ABS7.There is little nitrogenase activity in the nitrogen fixation area of nodule.Rhizobia is killed for plant immunity after entering nodule cells.Therefore,the mutant fn8013 is named nib2(nodules with impaired bacteroid).However,when inoculated with Rm1021 backgrounds of rhizobia,the nodule phenotype of fn8013 is slightly different.Rhizobia differentiate first after entering nodule cells,then die.3.After inoculated with three different rhizobia in green zeolite,the aboveground growth rate of fn9133 is inhibited.fn9133 form white columnar nodule.There is little nitrogenase activity in the nitrogen fixation area of nodule.Rhizobia differentiate first after entering nodule cells,then die.Further research show that nodule aged prematurely.Therefore,the mutant fn9133 is named debino3(decayed bacteroids in nodules).4.Through map based cloning,it is found that the nodule-deficient phenotype of mutant fn7852 is caused by Mt DMI1.In addition,the mutation site of nib2 is located within the region between molecular marker 005B12 and h2＿69d5a and the mutation site of debino3 is located within the region between molecular marker h2＿99d10d and 001H01.5.Through whole genome sequencing analysis,it is found that the candidate gene of nib2 is Mt NIB2.Online database analysis show that Mt NIB2 encodes a P-type phospholipid transporter(PLTP)and is highly expressed in root and nodules.After Mt NIB2-pro-CDS-p KGWRR is introduced by hairy root transformation,the transformed plants of nib2 return to wild-type phenotype.6.Through whole genome sequencing analysis,it is found that the candidate gene of debino3 is Mt DEBINO3.Online database analysis show that Mt DEBINO3 encodes a phosphoenolpyruvate carboxylase(PEPC)and is highly expressed in roots and nodules,specifically more expressed in nodules.After Mt DEBINO3-p KGWRR is introduced by hairy root transformation,the transformed plants of debino3 return to wild-type phenotype.In this study,two new mutants of nib2 and debino3 are found through phenotypic screening.The phenotypes of two mutants are elaborated preliminarily.The candidate genes of nib2 and debino3 are successfully found by the combination of map based cloning and whole genome sequencing.The genetic complementation of nib2 and debino3 is completed through hairy root transformation.It will provide a good research basis and ideas to study the functions of Mt NIB2 and Mt DEBINO3 in nitrogen fixation symbiotic metabolism. |
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