摘要： 为了研究拟南芥核酸酶AtCaN2基因在植物生长发育过程中的作用与功能，从拟南芥中克隆了核酸酶AtCaN2基因，并对该基因进行生物信息学分析，亚细胞定位分析，蛋白质诱导、表达、纯化及功能研究。结果表明，AtCaN2蛋白等电点为8.45，化学式为C₁₁₁₆H₁₇₈₈N₃₁₀O₃₃₅S₃，共含有3 552个原子数，为亲水性蛋白；AtCaN2与十字花科亚麻荠的同源性较高，同源性为87.89%，与谷子、高粱和玉米的同源性较低，同源性分别是56.42%，57.35%，56.47%，且在氨基酸序列中间具有高度保守的D-X-D序列；同时进化树分析显示AtCaN2与十字花科亚麻荠的亲缘性最近，与谷子、玉米等禾本科植物的亲缘性比较远，这些结果同氨基酸序列比对的结果一致；亚细胞定位结果显示其定位于细胞质，与预测结果相符；蛋白质诱导和纯化得到大小约为35 ku的His-AtCaN2融合蛋白；Western Blot分析显示，诱导表达的融合蛋白正确翻译表达，且没有出现结构上的断裂或者降解的情况；小量诱导和大量诱导均有点突变后的His-AtCaN2（M）融合蛋白表达，且纯化后得到了点突变His-AtCaN2（M）较单一的条带；蛋白酶活性分析表明，His-AtCaN2融合蛋白对核酸底物具有降解能力，表现出较高的活性，而His-AtCaN2（M）点突变融合蛋白对核酸底物没有降解能力，底物基本上未降解。研究结果可以为下一步在植物体内研究AtCaN2基因的相关机理作用提供基础。

关键词: 拟南芥, 核酸酶, AtCaN2, 亚细胞定位, 原核表达, 酶活性

Abstract: In order to study the role and function of the nuclease AtCaN2 gene in plant growth and development,the AtCaN2 gene was cloned from Arabidopsis thaliana. Bioinformatics analysis,subcellular localization analysis,protein induction,expression,purification and functional were carried out to study this gene. The results showed that the isoelectric point of AtCaN2 protein was 8.45,the chemical formula was C₁₁₁₆H₁₇₈₈N₃₁₀O₃₃₅S₃,which contained 3 552 atoms and it was a hydrophilic protein; AtCaN2 had high homology with Camelina sativa,the homology was 87.89%,and it had low homology with Setaria italic, Sorghum bicolor and Zea mays,the homology was 56.42%,57.35% and 56.47% respectively,among the amino acid sequences,there was a highly conserved D-X-D sequence; evolutionary tree analysis showed that AtCaN2 was closely related to C.sativa,but far from S.italic and Z.mays,whis were consistent with the results of amino acid sequence alignment; subcellular localization results showed that it was located in cytoplasm,which was consistent with the predicted results; the His-AtCaN2 fusion protein was induced and purified to be about 35 ku in size; Western Blot analysis showed that the fusion protein was correctly translated and expressed without structural breakage or degradation; the fusion protein of His-AtCaN2(M)was expressed in small amount and large amount of induction,and the single band of His-AtCaN2(M)was obtained after purification; enzyme activity analysis showed that His-AtCaN2 fusion protein had the ability to degrade the nucleic acid substrate,showing high activity,while the His-AtCaN2(M)point mutation fusion protein had no ability to degrade the nucleic acid substrate,and the substrate was basically not degraded.The results of this study provide a basis for further study of the related mechanism of AtCaN2 in plants.

Key words: Arabidopsis thaliana, Nuclease, AtCaN2, Subcellular localization, Prokaryotic expression, Enzyme activity

中图分类号: 

• Q78

• S55