RESUMEN
Not only calmodulin (CAM) with Ca2+ regulates the activity of many enzymes and proteins, but also free-CaM (no Ca2+bound) and Ca2+-independent CaM-binding proteins play roles in plant and animal cells. There is no in vivo method to identify the interaction between free-CaM and Ca2+-independent CaM-binding protein (CaMBP). Using site-directed mutagenesis by polymerase chain reaction (PCR), 5 mutant Arabidopsis calmodulin isoform 2 (AtCaM2) genes, mCaM21, mCoM212,mCaM2123, mCaM2124 and mCaM21234 were obtained. The mutant mCaM2 encoded glutamine in place of glutamate (E32Q; E68Q; E105Q; E141Q) in one or more EF-hand Ca2+-binding motifs of AtCaM2. The recombinant mCaM2 proteins were produced in Escherichia coli, and subsequently separated on SDS-PAGE in the presence of Ca2+ or EGTA, their electrophoresis mobilities were related with that of mutant EF-hand motifs. 45Ca2+ overlay analysis indicated that the more glutamate replaced by glutamine, the lower affinity with Ca2+ in the mCaM2 proteins. The mCaM21234 mutant protein (E32Q; E68Q; E105Q;E141Q) was unable to bind Ca2+. Using yeast two-hybrid technique with mCaM21234 as bait, it was possible to see interaction in Arabidopsis of AtCaM2 with IQD26, a calcium-independent CaM-binding protein. Site-directed mutation of AtCaM2 will aid the research of Ca2+, CaM and Ca2+-independent CaMBPs in plant biological processes.
RESUMEN
Genome of Arabidopsis has a kind of genes encoding proteins with ARM repeat domains and some of these proteins are known to play important roles in plant development and responses to hormone. An Arabidopsis mutant lfr with a distinct phenotype was got in leaf and flower development. The gene is predicted to encode a protein with ARM repeat domains. In order to study its function and molecular mechanism, recombination expression plasmid pGEX-2TGST∶LFR was constructed and transformed into the host bacteria strain Rosetta. Then IPTG was used to induce the recombinant protein expression in engineering strain. The expression products were detected by 12% SDS-PAGE. The GST∶LFR fusion protein was existed in soluble form with a relative molecular mass 77 ku, which is fit with the molecular mass supposed from gene coding frame. After purification by GST-tag affinity chromatography and electroelution, the fusion protein was used as antigen to prepare polyclonal antiserum in rabbits. After the fifth injection of antigen, the antiserum was obtained and further purified by decreased nonspecific bacteria and GST-tag antibody with method of immuno-precipitation. Western blot analysis showed that the purified antiserum, raised against the recombination LFR protein in rabbits, could react to the recombinant protein expressed in Rosetta specifically. And then the nuclear proteins of Arabidopsis wild type and mutant were extracted and separated by SDS-PAGE. Western blot assays revealed that there was a protein band, with a relative molecular mass 50 ku, indicating that antiserum could react to the native protein expressed in Arabidopsis specifically.