Construction Of Overexpression Plants And Mutants Of Lotus Japonicas Calmodulin Genes (LjCaMs)

Nitrogen is the life element of plants and animals,and it is an important and essential element in plant growth and development.The rational use of industrial nitrogen fertilizer could increase the yield and quality of crops,but the application of nitrogen fertilizer could not be completely absorbed by plants.Over-application resulted in eutrophication of soil and water;overuse of chemical fertilizers resulted in soil compaction and depletion,which leading to a decrease in crop yields,economic losses and environmental pollution.Nitrogen fixation symbiosis can not only satisfy the needs of plants for nitrogen elements,but also will not cause environmental pollution.Rhizobia can interact with leguminous plants,which is named nitrogen fixation symbiosis.The studies about molecular mechanism of nitrogen fixation symbiosis have achieved many achievements.In the nodulation signal pathway,calcium-calmodulin-dependent protein kinase(CCa MK)is used to decode Ca²⁺spiking.However,CCa MK only binding calmodulin(Ca M)can be activated to regulate the formation of effective nodules.In this study,Lotus japonicus calmodulin gene Lj Ca M2,Lj Ca M3 and Lj Ca M4 were selected as the objects.In order to explore the expression of Lj Ca M3 in different tissues,specific primers for 3'-RACE cloning were designed.q PCR was carried out.In addition,overexpression vector of p UB-GFP-Lj Ca M4 was constructed.The recombinant plasmids including p UB-GFP-Lj Ca M2,p UB-GFP-Lj Ca M3 and p UB-GFP-Lj Ca M4,were transferred into Agrobacterium rhizogenes AR1193 and Agrobacterium tumefaciens LBA4404.Hair roots transformation was carried out and transgenic plants were built.The ljcam2 and ljcam4 mutants were constructed using TILLING.These will provide experimental materials for the biological function studies of calmodulin genes.The main experimental results are as follows:1)Lj Ca M3 was cloned by 3'-RACE.And two fragments were obtained,which were637 bp and 749 bp named Lj Ca M3-1 and Lj Ca M3-2,respectively.In 3'-UTR region,Lj Ca M3-1 was 184 bp and Lj Ca M3-2 was 296 bp.Lj Ca M3-2 was 112 bp longer than Lj Ca M3-1,in which including 27.7%A,46.3%T,17%C and 9%G.Both Lj Ca M3-1 and Lj Ca M3-2 had 453 bp ORF,which encoded 150 amino acids.Its molecular formula is C₇₃₂H₁₁₅₇N₁₈₃O₂₅₇S₉;atomic number is 2338;relative molecular mass is 16921.85;isoelectric point(p I)is 3.96,and total average hydrophilicity is-.0411.In the secondary structure of Lj Ca M3,the?-helix accounted for 61%,the random coil accounted for 15%,the?-chain accounted for 1%;and it is a stable protein.2)The relative expression levels of Lj Ca M3 were tested by q PCR.There were significant differences between the different tissues.Roots and nodules had the highest expression levels,followed by stems>leaves>pods>flowers.3)Hair root transformations of Lj Ca M2,Lj Ca M3 and Lj Ca M4 were carried out.According to nodule phenotype,the hairy root-transformed plants of Lj Ca M2,Lj Ca M3 and Lj Ca M4 had significantly more nodules than that of Gifu after inoculation 11 day.4)The overexpression transgenic plants of Lj Ca M2,Lj Ca M3 and Lj Ca M4 were built.And three Lj Ca M2 T₀,four Lj Ca M3 T₀and two Lj Ca M4 T₀have been obtained.5)8 and 6 gene mutations of Ljcam2 and Ljcam4 were obtained by TILLING.For Ljcam2,only 4 mutations occurred in the coding region;and only one of them had a mutation in amino acid,which occurred at G⁸⁹⁹(G⁶⁰?D);For Ljcam4,only one mutations occurred in the coding region,which had a mutation in amino acid(E⁷⁴?K).This occurred at G⁴⁶⁵?A.After backcross with Gifu twice,five and two heterozygous mutant lines of Ljcam2 and Ljcam4 respectively were obtained.