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西洋参人参皂苷合成途径中原人参三醇合成酶基因CYP6H的克隆鉴定与功能分析
发表时间：2016-02-15 浏览量：2395 下载量：811
| 全部作者： | 王乐，赵寿经 |
| 作者单位： | 吉林大学生物与农业工程学院 |
| 摘 要： | 通过对比美国国立生物技术信息中心（National Center for Biotechnology Information，NCBI）已发表的人参原人参三醇合成酶基因CYP716A53v2 （GenBank登录号JX036031）和三七原人参三醇合成酶基因CYP450（GenBank登录号GU997670）的保守序列区设计简并引物，并对核心片段区序列进行cDNA末端快速扩增（rapid amplification of cDNA ends，RACE）的方式首次获得了西洋参中原人参三醇合成酶基因序列并命名为CYP6H. 对CYP6H进行氨基酸序列分析和亲缘关系树分析，表明CYP6H为P450基因家族中的一种且与原人参三醇合成酶基因具有极高的相似性和亲缘关系。进一步通过体外异源表达的方式进行功能鉴定分析，利用基因重组的方法构建重组表达载体pAUR123-CYP6H，转染酿酒酵母菌构建具有异源表达功能的重组酵母菌，向培养基中加入前体物质原人参二醇后，经高效液相色谱法（high performance liquid chromatography，HPLC）检测发现菌液提取物中含有催化产物原人参三醇，证明CYP6H为西洋参中的原人参三醇合成酶基因并发表于GeneBank（登录号：KC190491）. 以pBI121为载体，利用基因重组构建CYP6H的RNA干扰元件pBI121-CYP6H-RNAi和超表达元件pBI121-CYP6H-overexpression，通过转化发根农杆菌A4构建重组工程菌后侵染西洋参从而诱导西洋参发根体系的生成，HPLC检测发根中各种单体皂苷的含量变化用以进行CYP6H在人参皂苷合成途径中的功能分析。 |
| 关 键 词： | 生物工程；P450；异源表达；原人参三醇；功能分析 |
| Title： | Clone of protopanaxatriol synthase gene CYP6H during ginsenoside biosynthesis in Panax quinquefolius and functional analysis |
| Author： | WANG Le, ZHAO Shoujing |
| Organization： | College of Biological and Agricultural Engineering, Jilin University |
| Abstract： | Degenerate primers designed by comparing with conserved sequence region of the genes published on National Center for Biotechnology Information (NCBI), Panax ginseng CYP716A53v2 (GenBank number JX036031), P. notoginseng protopanaxatriol synthase gene CYP450 (GenBank number GU997670), we cloned a protopanaxatriol synthase gene CYP6H from P. quinquefolius for the first time by rapid amplification of cDNA ends (RACE). The P-BLAST search and phylogenetic tree revealed that CYP6H was a P450 gene and the deduced amino acid sequence of CYP6H shared extremely high similarity with other protopanaxatriol synthase. Identification of functional analysis was conducted by heterologous expression, using genetic recombination recombinant expression vector pAUR123-CYP6H, we transfected into Saccharomyces cerevisiae to construct recombinant yeast. Ectopic expression of CYP6H in recombinant S. cerevisiae resulted in the production of protopanaxatriol with added exogenous protopanaxadiol. The high performance liquid chromatography (HPLC) result revealed that CYP6H was protopanaxatriol synthase gene in P. quinquefolius (GenBank accession No. KC190491). RNA interferences component pBI121-CYP6H-RNAi and overexpression component pBI121-CYP6H-overexpression of CYP6H were constructed based on vector pBI121, then we converted Agrobacterium rhizogenes A4 recombinant engineering bacteria and infected P. quinquefolius to harvest P. quinquefolius hairy roots, ginsenoside content was analysed by HPLC to confirm the function of CYP6H in ginsenoside biosynthesis. |
| Key words： | bioengineering; P450; heterologous expression; protopanaxatriol; functional analysis |
| 发表期数： | 2016年2月第3期 |
| 引用格式： | 王乐，赵寿经. 西洋参人参皂苷合成途径中原人参三醇合成酶基因CYP6H的克隆鉴定与功能分析[J]. 中国科技论文在线精品论文，2016，9（3）：219-227. |
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