菠菜14-3-3蛋白基因的克隆及硝酸盐胁迫下的表达分析  

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

作　　者：赵秀玲[1] 徐慧妮[1] 郭传龙[1] 崔道雷[1] 王琳[1] 陈丽梅[1] 李昆志[1] 

机构地区：[1]昆明理工大学生命科学与技术学院,昆明650500

出　　处：《西北植物学报》2013年第3期450-457,共8页Acta Botanica Boreali-Occidentalia Sinica

基　　金：云南省中青年学术技术带头人后备人才培养基金(2006py01-10);云南省应用基础研究面上项目(2010ZC053);云南省教育厅科学研究基金(2001Z109);国家自然科学基金(31101557)

摘　　要：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 166bp,开放阅读框801bp,编码266个氨基酸。序列比对发现So14-3-3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56kD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。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.

关 键 词：菠菜 14-3-3 基因克隆 原核表达 硝酸盐胁迫 

分 类 号：Q786[生物学—分子生物学]