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作 者 ：孙静文， 周 卫， 梁国庆， 王秀斌

期 刊 ：植物营养与肥料学报 2011年 17卷 2期 页码：425-432

关键词：钙；钙调蛋白；Ca2 +-ATPase；基因表达；荧光定量PCR；苹果；

Keywords:Calcium, Calmodulin, Ca2 +-ATPase, Gene expression, Real-time PCR, Apple,

摘 要 ：研究不同浓度钙（0、1和10 mmo1/L CaCl₂ ；5 mmo1/L EGTA）对苹果果实钙调蛋白（CaM）含量和Ca²⁺-ATPase活性及其基因表达的影响。利用同源克隆方法分离CaM和Ca²⁺-ATPase基因，采用荧光定量PCR方法研究它们表达特征。结果表明，果实切片外源补钙，可溶性Ca²⁺及CaM含量在高钙处理12 h达到高峰；高钙处理12 h质膜Ca²⁺-ATPase活性显著增加，与胞内CaM含量增加时间一致；高钙处理24 h液泡膜Ca²⁺-ATPase活性显著增加；随着质膜和液泡膜Ca²⁺-ATPase活性显著增加，可溶性Ca²⁺含量在加钙处理48 h显著下降。研究基因表达发现，加钙处理6 h苹果CaM基因的表达量显著增加；加钙处理12 h苹果Ca²⁺-ATPase基因的表达量显著增加，与CaM含量及质膜Ca^{2 +}-ATPase活性变化一致。果实缺钙处理显著增加CaM基因表达量，而苹果Ca^{2 +}-ATPase基因的表达量没有显著变化。上述研究表明，苹果补钙可以提高细胞内可溶性Ca²⁺和CaM含量以及CaM基因的表达量，有效启动钙信使系统；质膜及液泡膜Ca²⁺-ATPase是调控胞内Ca²⁺关键的酶，通过提高质膜及液泡膜Ca²⁺-ATPase的活性及Ca²⁺-ATPase基因的表达量，维持胞内Ca²⁺的稳态水平。

Abstract:Effects of calcium on calmodulin (CaM) content, Ca^{2 +}-ATPase activity and their gene expressions in apple (Malus pumila) fruits were investigated under the hydroponic condition with 0，1，10 mmo1/L CaCl₂ and 5 mmo1/L EGTA. CaM and Ca^{2 +}-ATPase gene were isolated by homologous cloning and their expression patterns were analyzed by real–time PCR. Addition of 10 mmo1/LCaCl₂ to the culture solutions with 12 h exposure significantly increased soluble Ca^{2 +} and CaM contents in apple fruits. With the CaM content increasing, plasma and tonoplast membrane Ca^{2 +}-ATPase activities were enhanced after 12 and 24 h Ca exposure respectively, resulting in the decrease in the soluble Ca^{2 +} content after 48 h Ca exposure. In molecular level, the abundances of CaM and Ca^{2 +}- ATPase genes were up-regulated after 6 h and 12 h Ca exposure, respectively, in accordance with change of the CaM content and plasma membrane Ca^{2 +}-ATPaseactivity. Addition of 5 mmo1/L EGTA to the culture solutions induced up-regulated the expression of the CaM gene, however, did not markedly change the expression of the Ca^{2 +}-ATPase gene. It was concluded that external calcium might increase soluble Ca^{2 +} and CaM contents and the expression of the CaM gene to stimulate an intracellular Ca^{2 +}/calmodulin signaling system. Plasma and tonoplast membrane Ca^{2 +}-ATPase are of major physiological importance to expel Ca^{2 +} from eukaryotic cells and to maintain overall Ca^{2 +} homoeostasis by enhancing their activities and gene expression.

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