The Identification Of Calmodulin Gene In Lotus Japonicas And The Studies Of The Expression And The Activity Of LjCaM4

Nitrogen fertilizer is the necessary nutrients for crops growth and enhancement of yield and quality,but it cannot be completely absorbed by plant,which results in seriously soil salinization and water eutrophication.The nitrogen fixation symbiosis between legume and Rhizobium can transfer the nitrogen in the air into the form that plants can absorb and use,which plays an important role in the sustainable development of environment and agriculture.The studies on the molecular mechanism of root nodule symbiosis have made some achievements.It was found that the response of host plant cells to the early stage of Rhizobium infection was Ca2+ spiking.Calcium-and calmodulin-dependent protein kinase(CCaMK)which plays a key role in both infection thread formation and nodule primordia organogenesis can decode the Ca2+ signal,in order to activate the signal transduction cascade of events.The studies found that binding Ca2+ to CCaMK can regulate the auto-inhibition to release,which plays a key role in nodule primordia organogenesis;and CCaMK would show the kinase activity when there was calmodulin(CaM),which plays a key role in infection thread formation.In this study,the phylogenetic tree was built and the calmodulin genes in Lotus japonicus(Regel)K.Larsen were identified.In order to explore the expression and the activity of LjCaM4,the 3'-RACE clone primers of LjCaM4 were designed.The qPCR was used to analyze the expression of LjCaM4 in different tissues.Using pET28-a(+),the prokaryotic expression vector was constructed and expressed in Rosetta;The LjCaM4 protein was purified by Ni Sepharose TM affinity chromatography column,and then the binding ability to Ca2+ and mobility were analyzed by SDS-PAGE.The main results are as follows:1)In order to identify EF-hand-containing proteins,the L.japonicus genome available in the Kazusa Resources for Interpro Database Matches was searched using three different methods,HMMPfam,HMMSmart and Superfamily.Second,the L.japonicus database was searched with AtCaM1 as the query amino acid sequence using the program BLASTp,and the unfound five sequences(E-value>3e-64)were added to the list.Each identified amino acid sequence was analyzed for EF hands motifs and other functional domains using the InterProScan software using the default settings.A total of 47 putative proteins were identified that do not contain any other identifiable domains other than the Ca2+-binding domain in Lotus.The amino acids in the identified proteins are 23%identical with LjCaM1 and were analyzed further.ML and BI analyses produced major rule consensus trees with identical topologies except for the different BI posterior probability values and ML bootstrap values.The 26 genes were separated into two groups.Group I consisted of seven genes called calmodulins(LjCaMs)and the rest of the 19 genes from phylogenetic tree formed group ? called calmodulin like proteins(LjCMLs).2)The LjCaM4 gene was cloned by 3'-RACE,and the 642 bp sequence including the complete ORF and PolyA was obtained.The biological information analyses found that the molecular formula of LjCaM4 protein is C741H1168N186O260S9 and the atomic number is 2364;the molecular weight is 17.13 kDa;the theoretical pI value is 4.03;the predicted half-life is 30 h;the instability coefficient is 35.46;the average hydrophilicity was-0.505.In the secondary structure of LjCaM4 protein,a-helix accounts for 65.33%;random coil accounts for 23.33%;Extending chain accounts for 6%;P-bend accounts for 5.33%.The secondary structure was rich in alpha helix.LjCaM4 protein has no transmembrane region,and the 4 EF hands are located in 12-40,48-76,85-113,and 121-149,respectively.And there are 16 Ca2+ binding sites.3)To detect the expression of LjCaM4 in different tissues of L.japonicas,the qPCR was carried out.The expression of LjCaM4 in different tissues are significantly different and the highest expression level is in the roots.The expression level is roots>stem>leaf>pods>flower.4)After BamH I and Xho I digestion,the LjCaM4 was recombined into pET28-a(+),and then the recombinant plasmid pET28-a(+)-LjCaM4 transfer into Rosetta.The positive clones were screened by antibiotic and PCR.The induced expression were carried out under different IPTG concentration(1 mmol/L,2 mmol/L and 3 mmol/L)at 37?.The supernatant and precipitation were analyzed by SDS-PAGE electrophoresis.And there was a specific band at the size of 20.1 kDa,indicating that the protein expression was obtained.The supernatant was purified by Ni Sepharose TM and the total concentration of the purified protein was determined by BCA assay.The concentration was 360 ?g/mL.5)Using EGTA as the calcium chelator and LjCaM1 protein as control,LjCaM1 and LjCaM4 protein in Ca2+ were compared respectively with the electrophoretic mobility and after treatment with EGTA,the results indicated that differences in electrophoretic mobility between Ca2+ and EGTA after the treatment of LjCaMl and LjCaM4 protein,LjCaM4 is 2 times of LjCaM1 protein electrophoresis mobility change.The results can provide a theoretical basis for further studies of biological function of LjCaM4.