Site-directed Mutagenesis of Arabidopsis Calmodulin Isoform 2 and Its Application in Detecting Calcium-independent Calmodulin-binding Proteins
10.3724/SP.J.1206.2008.00786
- VernacularTitle:拟南芥钙调素定点突变基因分离及其在钙不依赖钙调素结合蛋白检测中的应用
- Author:
Li GAO
;
Zhenjie WANG
;
Sujuan CUI
- Publication Type:Journal Article
- Keywords:
site-directed mutagenesis;
Arabidopsis;
calmodulin;
calcium-independent;
calmodulin-binding protein
- From:
Progress in Biochemistry and Biophysics
2009;36(7):890-896
- CountryChina
- Language:Chinese
-
Abstract:
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.
