菠菜14-3-3蛋白基因的克隆及硝酸盐胁迫下的表达分析

利用RT-PCR和RACE技术,从菠菜中首次获得了14-3-3蛋白基因的全长cDNA序列(GenBank登录号JX952165),命名为So14-3-3.该基因全长1 166 bp,开放阅读框801 bp,编码266个氨基酸.序列比对发现So143 3蛋白与其他植物14-3-3蛋白氨基酸序列一致性高达77.6％～84.7％.半定量RT-PCR表明,随NO3-胁迫处理时间的延长和浓度的增加,菠菜根和叶中So14-3-3基因的表达增强.实验构建了pGEX4T-So14-3-3原核表达载体,并通过IPTG诱导后获得分子量约为56 kD的蛋白.进一步的蛋白质印迹检测结果表明,随着NO3处理时间的延长和浓度的增加,So14-3-3蛋白表达也增加.该实验结果为进一步研究So14 3-3蛋白功能提供了基本的实验基础.

Cloning and Expression Analysis of 14-3-3 Protein Gene of Spinach under Nitrate Stress

Using RT-PCR and RACE,we obtained the spinach 14-3-3 protein gene full-length cDNA sequence (GenBank accession No.JX952165),and designated So14-3-3.The full length sequence was 1 166 bp,with the ORF of 801 bp,which encodes 266 amino acids.Amino sequence alignment showed that So14-3-3 had high identity with other plants 14-3-3.The expression of So14-3-3 in root and leaf increased with the increasing nitrate treatment time and NO₃^- concentration by RT-PCR.The prokaryotic expression vector pGEX4T-So14-3-3 was constructed.A recombinant protein induced by IPTG with a molecular weight of 56kD was obtained.Further,Western blotting analysis showed that the expression of So14-3-3 also increased with the increasing nitrate treatment time and NO₃^- concentration.These results provide experimental base to further study the function of So14-3-3.