| 拟南芥AtCOR15a抗寒基因原核表达载体的构建及蛋白表达 |
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| 引用本文： | 李静,李晓荣,王冬梅,郝晓燕,李建平,黄全生,张桦.拟南芥AtCOR15a抗寒基因原核表达载体的构建及蛋白表达[J].新疆农业科学,2010,47(10). |
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| 作者姓名： | 李静 李晓荣 王冬梅 郝晓燕 李建平 黄全生 张桦 |
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| 基金项目： | 新疆维吾尔自治区重大专项 |
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| 摘 要： | 【目的】通过RT-PCR获得拟南芥AtCOR15a基因的ORF全长,经酶切、连接,以pET-28a(+)为原核表达载体。构建成pET28a-COR15a的重组表达载体,研究该基因在大肠杆菌中的表达情况,为进一步研究该基因的功能奠定基础。【方法】根据拟南芥AtCOR15a基因序列设计特异引物,利用RT-PCR的方法扩增目的基因,构建原核表达载体pET28a-COR15a,并将此重组质粒转化到大肠杆菌菌株BL21(DE3)中,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,SDS-PAGE电泳分析,验证其蛋白表达量,并对其体系进行优化。【结果】AtCOR15a在大肠杆菌中成功表达,在37℃条件下诱导表达量最大,而30和16℃条件下蛋白表达量降低,IPTG浓度0.2~1.0 mmol/L对蛋白的表达量影响不大,并没有明显差异。但通过在不同诱导时间取样分析,结果发现加入IPTG后诱导5 h蛋白表达量最大,而8 h后逐渐降低。【结论】构建的原核表达载体在大肠杆菌中能高效表达,AtCOR15a融合蛋白分子量大约为19 kD,为进一步研究该基因的功能奠定了基础。
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| 关 键 词： | AtCOR15a 原核表达载体 蛋白表达 |
Construction of Prokaryotic Expression Vector and Protein Expression in Cold Resistance gene AtCOR15a of Arabidopsis Thaliana |
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| LI Jing,LI Xiao-rong,WANG Dong-mei,HAO Xiao-yan,LI Jian-pin,HUANG Quan-sheng,ZHANG Hua.Construction of Prokaryotic Expression Vector and Protein Expression in Cold Resistance gene AtCOR15a of Arabidopsis Thaliana[J].Xinjiang Agricultural Sciences,2010,47(10). |
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| Authors: | LI Jing LI Xiao-rong WANG Dong-mei HAO Xiao-yan LI Jian-pin HUANG Quan-sheng ZHANG Hua |
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| Abstract: | 【Objective】By RT-PCR of Arabidopsis thaliana,AtCOR15a ORF complete gene length were obtained,after restriction enzyme and connection,and the pET28a-COR15a recombinant expression vector from pET-28a(+)prokaryotic expression vector were constructed to study the recombinant gene expression in E.coli.【Method】According to the Arabidopsis AtCOR15a gene sequence,specific primers were designed.The target gene was amplified by RT-PCR,then the prokaryotic expression vector pET28a-COR15a were constructed,and this recombinant plasmid got was transformed into BL21(DE3),after isopropyl-β-D-thiogalactopyranoside(IPTG)inducing expression,then SDS-PAGE electrophoresis were made to verify the protein expression,and optimize the system.【Result】AtCOR15a gene was successfully expressed in E.coli.At 37℃,induced expression level is the largest,while under the conditions of the 30℃ and 16℃,much lower proteins were expressed,IPTG concentration from 0.2 mmol/to 1.0 mmol/L has little effect on the protein expression without significant difference.By sample analysis at different time of induction production,it was found that the greatest induction protein was expressed after adding IPTG to system through reaction for the 5 h,but the protein yield gradually was decreased after 8h.【Conclusion】The recombinant AtCOR15a gene inserted prokaryotic expression vector can be efficiently expressed in E.coli,a fusion protein molecular weight of approximately 19 kD was produced.This thesis would lay a foundation for the further study of physical and chemical properties of the fusion protein. |
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| Keywords: | AtCOR15a prokaryotic expression vector protein expression |
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