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

动植物系统研究表明，钙调素不仅在结合钙离子时调节多种靶酶或靶蛋白的活性，而且没有钙离子结合时，还可以通过结合钙不依赖的钙调素结合蛋白，发挥多种生物学作用．然而，目前却没有体内分析钙调素与钙不依赖钙调素结合蛋白相互作用的方法．首先，采用定点突变的方式，得到了拟南芥钙调素亚型2的多个突变基因mCaM2，随后，大肠杆菌重组表达突变蛋白的电泳迁移率及⁴⁵Ca²⁺覆盖分析表明，得到了编码失去钙结合能力的钙调素的突变基因mCaM2₁₂₃₄, mCaM2₁₂₃₄突变钙调素中所有4个钙结合EF-hand结构域中的关键氨基酸谷氨酸均突变为谷氨酰胺．在酵母双杂交体系中，作为诱饵蛋白的突变钙调素mCaM2₁₂₃₄与我们前期体外方法报道的钙不依赖性钙调素结合蛋白AtIQD26存在相互作用．这将为钙不依赖性钙调素结合蛋白提供有用的体内研究工具，有利于我们全面认识钙-钙调素-钙调素结合蛋白信号途径．

Site-directed mutagenesis of Arabidopsis calmodulin isoform 2 and its application in detecting calcium-independent calmodulin-binding proteins

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, mCaM2₁₂, 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.