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黄芩是一种传统的中药,黄芩苷是其重要的药用成分之一,具有广泛的药理作用并被用于多种疾病的治疗,具有巨大的药用价值和经济价值。CHS是催化黄芩苷合成限速步骤的关键酶,其表达量对黄芩苷的积累起关键的作用。本研究利用基因工程的手段,构建黄芩的CHS过表达载体,并将其导入根癌农杆菌中,拟在黄芩中持续表达CHS,促进黄芩苷合成。基于NCBI上公布的黄芩基因信息,设计特异引物,通过RT-PCR技术扩增CHS的c DNA全长序列,并利用生物信息学软件对序列进行分析,在引物两端引入特异酶切位点,将目的序列CHS通过限制性内切酶酶切和连接的方式,构建载体p BI 121-35S-CHS。通过PCR和酶切的方法进行验证,获得黄芩CHS基因的高效表达载体,可直接用于黄芩的遗传转化,使CHS基因表达量增加,促进黄芩苷的合成。
Scutellaria baicalensis is a traditional Chinese medicine, baicalin is one of its important medicinal ingredients, has a wide range of pharmacological effects and is used for the treatment of a variety of diseases, with great medicinal value and economic value. CHS is a key enzyme that catalyzes the rate-limiting step of baicalin synthesis, and its expression level plays a key role in the accumulation of baicalin. In this study, we constructed the CHS overexpression vector of Scutellaria baicalensis Georgi by genetic engineering and introduced it into Agrobacterium tumefaciens, and it is intended to continuously express CHS in Scutellaria baicalensis and promote the synthesis of baicalin. Based on the published information of Scutellaria baicalensis Georgi on the NCBI, specific primers were designed and the full-length cDNA of CHS was amplified by RT-PCR. Bioinformatics software was used to analyze the sequences and specific restriction sites were introduced at both ends of the primers. The vector p BI 121-35S-CHS was constructed by digesting and ligating the target sequence CHS by restriction enzyme. Through PCR and enzyme digestion, we obtained the efficient expression vector of CHS gene of Scutellaria baicalensis Georgi, which can be directly used for the genetic transformation of Scutellaria baicalensis Georgi and increase the expression of CHS gene to promote the synthesis of baicalin.