In Vitro Activity Assay Of Calmodulins(LjCaMs) In Lotus Japonicus

Nitrogen is a necessary element of living organisms for the growth and development.Artificial nitrogen fixation caused a lots of environmental problems.In contrast,biological nitrogen fixation is more efficient and environmentally friendly.Nitrogen fixation symbiosis?NFS?is the most important and prominent biological nitrogen fixation in leguminous plants.It has been found that Ca²⁺spiking would be caused after bacteria infected host plants.And this signals generated by Ca²⁺spiking needs to be decoded by calcium-and calmodulin-dependent protein kinase?CCaMK?.Only when the concentration of Ca²⁺in the body changes in the presence of calmodulin?CaM?,CCaMK can be activated and participated in the regulation of NFS.The CaMs family is widely present in plants and encode different isoforms.Studies have shown that different CaMs isoforms have different biological properties and show different biological functions.Therefore,in this study,the CaM proteins of Lotus japonicus?Regel?K.Larsen were studied to explore the diversity of their biological activities in vitro.The main experimental results are as follows:1)CCaMK and ccamk-14 were obtained by site-directed mutagenesis,and then the prokaryotic expression vectors were constructed.The expressions were induced at 28°C with 1 mmol/L IPTG for 8 h.It were found that there were specific protein bands?about 57kDa?in the the precipitation at 28°C,suggesting that they were successfully expressed.The purified protein concentrations were determined by BCA kit.CCaMK was 415.1 g/mL and CCaMK^S337N was 474.7 g/mL.2)pET28-a?+?-LjCaMs were expressed at 37°C,9h,1 mmol/L IPTG and purified.The concentrations of EGTA complete eluting different LjCaMs and the binding abilities of LjCaMs to Ca²⁺were all different.When the purified LjCaMs were 9?g and the reaction system was 50?L,it was found that LjCaM1 and LjCaM3 could be completely eluted by0.1?mol EGTA,LjCaM2 could be completely eluted by 0.05?mol EGTA,and LjCaM4could be completely eluted by 0.2?mol EGTA.LjCaM1 was saturated when the Ca²⁺was1.25?mol;LjCaM2 was saturated at 1.4?mol;LjCaM3 was not saturated at 2.5?mol,and LjCaM4 was saturated at 2?mol.Therefore,the binding abilities of LjCaMs to Ca²⁺was LjCaM3>LjCaM4>LjCaM2>LjCaM1.3)The interaction between LjCaMs and CCaMK/CCaMK^S337N337N were detected in vitro.The results showed that CCaMK/CCaMK^S337N expressed in prokaryotic cells could not directly interact with LjCaMs.It was speculated that the Ca²⁺concentrations in the reaction buffer were insufficient and CCaMK was not phosphorylated.And further experiments will need to be done with increasing the Ca²⁺concentration in the reaction buffer and phosphorylated CCaMK.4)The BiFC of LjCaMs with CCaMK and ccamk-14 were performed in protoplasts of Arabidopsis protoplasts.It was proved that LjCaMs could interact with CCaMK/CCaMK^S337N.The interaction between LjCaM1 and CCaMK mainly occurred in the plasma membrane and the cytoplasm;the interaction between LjCaM2 and CCaMK occurred in the plasma membrane,the chloroplast membrane,the nucleus and the cytoplasm;the interaction between LjCaM3 and CCaMK occurred in the plasma membrane,the chloroplast membrane and the cytoplasm;the interaction between LjCaM4 and CCaMK mainly occurred in the plasma membrane;the interaction between LjCaM1/4 and CCaMK^S337N mainly occurred in the plasma membrane;the interaction between LjCaM2and CCaMK^S337N occurred in the plasma membrane,the chloroplast membrane and the cytoplasm;the interaction between LjCaM3 and CCaMK^S337N mainly occurred in the plasma membrane,the cytoplasm and the nucleus.This suggests that mutation in CCaMK affects its binding to CaM.