Study On The Clone And Expression Of Ca²⁺/CaM Dependent Protein Kinase Genes Of Magnaporthe Grisea

In this dissertation,Ca²⁺/calmodulin-dependent protein kinase(CaMK)genes of Magnaporthe grisea,named MgCaMK1(GeneBank no.EU 545466)and MgCaMK2 (GeneBank no.EU545467),respectively,were cloned.Also,the expressing characteric of two genes in bacteria and the kinase activity of expressing products were studied.The main results were as follows:(1)The MgCaMK1 cDNA was composed of 1221 nucleotides,including 5 introns, with an open reading frame encoding 406 amino acid residues and the molecular weight was 45.6 KDa.MgCaMK2 cDNA was composed of 1260 nucleotides, including one intron,with an open reading frame encoding 419 amino acid residues and the molecular weight was 45.7 KDa.The sequence of encoding proteins of MgCaMK1 or MgCaMK2 showed homology to other CaMKs of fungi.The sequence identity between MgCaMK1 and CaMKs in Neurospora crassa or Sporothrix schenckii was 76%.The MgCaMK2 sequence showed 88%identity to CaMK of Aspergillus clavatus.The phylogenetic tree analysis basing on the amino acid sequences suggested that MgCaMK1 or MgCaMK2 showed closer relative relations to other CaMKs from different species.(2)Southern blot analysis indicated that MgCaMK1 or MgCaMK2 gene was single copy in Magnaporthe grisea genome.(3)The expression of MgCaMK1 or MgCaMK2 in Yeast Expressing Systerm was not successful.So,the expression of MgCaMK1 or MgCaMK2 in E.coli BL21 by virtue of pET-32a(+)plasmid was prosecuted.The inductive conditions to produce more soluble fusion proteins were obtained.Under the conditions with a concentration of 1.0 mM IPTG,20 hours inductive time,15℃inductive temperature,the amount of soluble Trx-MgCaMK1 was more than 64.8%.Under the conditions with a concentration of 0.8 mM IPTG,20 hours inductive time,15℃inductive temperature, the amount of soluble Trx-MgCaMK2 was more than 39.1%.(4)The autophosphorylation and substrate phosphorylation activity analysis to both of the pure expressed products showed that MgCaMK1 and MgCaMK2 could phosphorylate itself and Histoneâ…¢-s.However,the activity of MgCaMK1 exhibited a calcium/calmodulin-dependent manner,and this result suggested that MgCaMK1 is a kind of calcium/calmodulin-dependent protein kinase.The activity of MgCaMK2 only displayed a calcium-dependent manner but not calmodulin-dependent manner,this result implyed that MgCaMK2 might be a kind of calcium-dependent protein kinase.