柴胡MVA通路中IPPI基因在大肠杆菌中的克隆柴胡为伞形科植物，性味苦，微寒。归肝经、胆经。按性状不同，分别称北柴胡及南柴胡。本文是以北柴胡作为研究对象。北柴胡的有效成分主要是柴胡皂苷，是一种齐墩果烷类型的三萜皂苷，具有抗内毒素作用。通过以柴胡皂苷合成的途径作为前提，将其中的关键酶ippi提取出来后，进行cDNA的克隆，得知基因序列后，设计一对引物。本文以北柴胡根作为原料，提取RNA，对RNA进行反转录得到cDNA，通过引物对cDNA进行PCR的扩增得到异戊烯基焦磷酸异构酶基因片段，大小为532bp，编码是177，再通过对质粒的提取并连接后，将重组子转入大肠杆菌中，通过双酶切检测是否克隆成功。每完成一步操作都需要通过琼脂糖凝胶电泳来检测是否提取成功，从而达到分离、鉴定分子量，筛选重组子的目的。甲羟戊酸属于一种代谢途径，以coA为原料，通过不同反应，合成IPP和DMAPP。而关键酶IPPI有重要的作用。而此途径在大肠杆菌中未有转化。本文将研究北柴胡中柴胡皂苷的关键酶基因IPPI的cDNA克隆，为研究调控柴胡皂苷类的生物合成提供了基础。
关键词：柴胡皂苷；甲羟戊酸；PCR；异戊烯基焦磷酸异构酶；大肠杆菌
Abstract: Bupleurum is an umbelliferae plant with bitter taste and mild cold. Return to liver and bile. According to different traits, they are respectively called North Bupleurum and South Bupleurum .This paper is a research object of North Bupleurum. The mainly active component of Bupleurum is Radix Stellaviae, which is a triterpene saponin of the bupleurum type and has an anti-endotoxin effect. By cloning isopentenyl pyrophosphate isomerase cDNA, one of the key genes in the northern radix stellaviae synthesis pathway, the sequence of this gene fragment was obtained, which laid the foundation for further understanding the regulation of radix stellaviae's biosynthesis.In this paper, the total RNA of the young roots of bupleurum chinensis translate RNA to the cDNA. By increasing the cDNA with a primer, the isoprene pyrophosphate isomerase gene fragment is obtained, and the size is 532bp. The encoding is 177. After the Plasmid is extracted and connected, the recombinant is transferred to E. coli, and whether the cloning is successful is detected by double enzyme cutting. Each step of the operation needs to be tested by agarose gel electrophoresis to obtain the success of the extraction, so as to separate and identify the molecular weight and screen the recombinant.Because methylvalproic acid is a metabolic pathway of isoprene pyrophosphate and dimethylallyl pyrophosphate synthesized from acetyl-CoA, isopentyl pyrophosphate isomerase(IPPI) is one of the key enzymesthe in main route which in the methovalonic acid pathway. Bupleurum's methyvalic acid pathway was not converted in E. coli. In this paper,we are studied the cDNA cloning of the key enzyme gene IPPI in the root of the bupleurum chinensis ,which provided a basis for the study of the biological synthesis of bupleurum chinensis.
Key words: Radix Stellaviae;mevalonic acid; PCR; IPPI;Escherichia coli
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