Study On The Function Of Lotus Japonicus Carbonic Anhydrase Gene In Symbiotic Nitrogen Fixation

Carbonic anhydrase?Carbonic anhydrases,CA;EC 4.2.1.1?is widely found in nature and has been identified in microorganisms,animals and plants.CA is a metalloenzyme that catalyzes the reversible reaction of CO₂ and HCO₃^-in the organisms:CO₂+H₂O?HCO₃^-+H⁺,which is important for the normal growth and metabolism of organisms.The presence of CA greatly accelerates the rapid progression of this reaction.At present,six types of CA genes have been found in nature,namely:?-,?-,?-,?-,?-,and?-CA.The main CA in plants are?-,?-,and?-CAs.Moreover,most of the CA genes in plants are polygenic,and the location and expression level of CA in the same organism are also different due to their respective functions.CA found in plants play a certain role in photosynthesis,biological stress,seed development,p H regulation,biosynthesis and so on.CA have also been identified in legumes such as alfalfa,soybean and Lotus japonicus,but the role of CA in symbiotic nitrogen fixation of legumes and rhizobia has not been identified.In this study,we analyzed the evolution,expression pattern and mutant phenotype of CA in the model legume,Lotus japonicus.The main experimental results obtained so far are as follows:1. Taking the CAs in Arabidopsis as the template,blast sequence alignment was carried out in the Lotus japonicus database website,all CAs from Lotus japonicus were found,and the evolutionary tree was constructed together with the CAs from Arabidopsis.It was found that CAs belonged to the polygenic family in Lotus japonicus.There are 12CAs genes:6 encode?-CAs,4 encode?-CAs and 2 encode?-CAs.evolutionarily,?-CAs and?-CAs are closely related.2. CAs in Lotus japonicus have different expression patterns,Ljcaa1 only expressed in the root nodule,Ljcaa2 expressed in all tissues except roots,and the highest expression was found in seedlings,Lj5g3v0670540.1 expressed in roots,seedlings and nodules,Lj5g3v0670150.1 expressed in roots,flowers,fruit pods,seedlings,but not in nodules.Lj3g3v3082370.1 is only weakly expressed in the roots.Lj CA1 expressed in all tissues of Lotus japonicus,and it is most expressed in root nodules.Lj6g3v2193530.1 weakly expressed in tissues other than root nodules,Lj2g3v1002750.2 only expressed in above-ground tissues,with the highest expression in leaves and cotyledons.Lj2g3v1403790.1 expressed in all tissues,with the highest expression in cotyledons.Lj CAA1 expressed in both infected and uninfected cells,Lj CAA2 expressed only in nodule vascular bundles,and Lj CA1 expressed in uninfected cells.3. It was found that the single CA gene knockout did not affect the symbiotic nitrogen fixation of Lotus japonicus.All the homozygous single mutants of ca1,caa1,and caa3,and the homozygous double mutants of caa1caa3 could perform nitrogen-fixing function normally,and the homozygous overexpressing plants of Lj CA1 showed no significant difference to the wild-type phenotype.In summary,this subject systematically studied the carbonic anhydrase gene in Lotus japonicus,and learned the classification of the carbonic anhydrase gene in Lotus japonicus;the expression of carbonic anhydrase in the tissues of Lotus japonicus.Successfully constructed homozygous ca1 single mutant,caa1 single mutant,caa3 single mutant and caa1caa3 double mutant,we also performed functional prediction of carbonic anhydrase genes in Lotus japonicus.These work laid a solid foundation for further study of carbonic anhydrase gene.