| 摘要: |
| 【目的】克隆人参皂苷生物合成途径中的鲨烯环氧酶（SQE）基因，并进行原核表达与纯化，初步探讨SQE活性与人参皂苷生成量之间的关系。【方法】以4年生人参根组织须根为材料，提取其总RNA，反转录为cDNA。以合成的cDNA为模板，对SQE基因进行克隆，再将其插入原核表达载体pET-30a中，构建pET-30a-SQE重组质粒，经酶切和测序鉴定后，转入Rosetta大肠杆菌，经0.8 mmol/L IPTG 37 ℃诱导表达4 h后，进行SDS-PAGE电泳检测，采用Ni Agarose亲和层析柱纯化目的蛋白，利用液相色谱 串联质谱联用技术（LC-MS）检测SQE的活性。【结果】获得了人参SQE基因1 611 bp的全长编码区cDNA。酶切和测序结果表明，原核表达载体pET-30a-SQE构建成功；SDS-PAGE分析显示，在Rosetta大肠杆菌中成功诱导表达了SQE融合蛋白，且纯化后的目的蛋白纯度较高；LC-MS联用检测结果发现，随着SQE用量的增加，达玛烯二醇的生成量递增。【结论】克隆了人参SQE基因，获得了在体外具有生物学活性的SQE蛋白，并证实了其活性与人参皂苷生成量有很大的相关性。 |
| 关键词: 人参 鲨烯环氧酶 cDNA克隆 原核表达 |
| DOI： |
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| 基金项目:国家自然科学基金项目（30570185，31070316） |
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| Cloning,expression of squalene epoxidase from Panax ginseng |
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| Abstract: |
| 【Objective】The research was conducted to determine the relationships with enzyme activity of squalene epoxidase (SQE) and the accumulation of ginsenosides after cloning and expression a full-length cDNA encoding squalene epoxidase for the biosynthetic pathway of ginsenosides.【Method】Fouryear ginseng root tissue fibrous was used as material.After extracting the total RNA and reversing transcription,primers were designed and then SQE gene cloned.And the production was inserted to expression vector pET-30a to obtain recombinant plasmid pET-30a-SQE and expressed (0.8 mmol/L IPTG for 4 h at 37 ℃) in the host cells Rosetta.The results were analysed by SDS-PAGE.The recombinant protein was purifed by affinity chromatograph.And the enzyme activity of SQE was determined with LC-MS.【Result】The results showed that complete encoding sequence of SQE in ginseng root was 1 611 bp,which encoded 537 amino acid residues.Restriction enzyme mapping and sequencing showed that pET-30a-SQE expression vector was constructed successfully.SDS-PAGE showed that fusion protein of SQE was induced in Rosetta host cells and the purity of interest protein was very high.The production of dammarendiol was increased gradually as the concentration of SQE enzyme increased after liquid chromatography tandem mass spectrometry (LC-MS) analysis.【Conclusion】SQE gene was cloned and expressed,and gained active enzyme of SQE in vitro.The results indicated that there were significant correlations between the enzyme of SQE and the contents of total ginsenosides. |
| Key words: Panax ginseng squalene epoxidase cDNA cloning prokaryotic expression |
