| 摘要: |
| 【目的】克隆大豆查尔酮还原酶3基因（chr3），并分析其编码蛋白体外催化活性，为研究大豆苷元的合成与调控研究提供参考。【方法】以大豆品种“吉农17”为材料，采用RACE技术扩增大豆chr3基因，对其编码蛋白的结构和功能位点进行分析。将目的基因chr3连接到大肠杆菌载体pET28a上，然后转化到大肠杆菌BL21中，构建重组大肠杆菌原核表达载体BL21-pET28a-chr3，并通过高效液相色谱技术（HPLC）验证重组蛋白的催化性质及效率。【结果】成功克隆chr3基因，其全长序列长1 416 bp（GenBank 登录号:KF927169），成功构建了大肠杆菌原核表达载体BL21-pET28a-chr3。HPLC检测结果表明，重组蛋白CHR3催化合成了异甘草素，使大豆异甘草素含量提高到了33.673 μmol/g。【结论】分离鉴定了大豆chr3，其编码蛋白CHR3催化活性明显提高。 |
| 关键词: 大豆 大豆苷元 查尔酮还原酶3基因 基因克隆 大肠杆菌 |
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| 基金项目:国家自然科学基金面上项目“大豆查尔酮还原酶（CHR）在大豆苷元合成中的作用机理”（31171568） |
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| Cloning of soybean chalcone reductase gene (chr3) and its expression in Escherichia coli |
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ZHANG Chao,WANG Pi-wu,ZHANG Zhuo
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| Abstract: |
| 【Objective】Soybean chalcone reductase 3 (chr3) was cloned and its coding protein catalytic in vitro was analyzed to provide references for synthesis and regulation of daidzein.【Method】The soybean chr3 was amplified by RACE technology and the domain and function of its coding protein was analyzed using soybean “Jinong 17” as material.The fragment was introduced into pET28a vector,which was transformed into E.coli BL21.Recombinant vector BL21-pET28a-chr3 was constructed and recombinant protein was verified by high performance liquid chromatography (HPLC).【Result】The genes chr3 (GenBank accession:KF927169) with full length of 1 416 bp was cloned successfully.The recombinant vector BL21-pET28a-chr3 was constructed successfully,and HPLC showed that synthesis of isoliquiritigenin catalyzed by recombinant protein increased the content of soybean isoliquiritigenin to 33.673 μmol/g.【Conclusion】The soybean chr3 genes was isolated and identified,which improved catalytic activities of coding protein CHR3. |
| Key words: soybean daidzein chalcone redutase 3 gene (chr3) gene cloning Escherichia coli |
