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目的:获得粘毛黄芩查尔酮合成酶(CHS)基因正反义植物表达载体,通过转化粘毛黄芩进一步探讨CHS在黄酮化合物生物合成途径上的作用机理。方法:通过RT-PCR从粘毛黄芩总RNA扩增出CHS基因目的片段,克隆至PMD18-T载体,测序并进行生物信息学分析。结果:测序证明序列正确,同时推测出CHS氨基酸序列及三维结构。克隆片段和pCAMBIA1304+载体用BlgII和BstE II双酶切,然后连接CHS基因目的片段及pCAM-BIA1304+载体大骨架片段,得到CHS正反义植物表达载体。结论:成功构建粘毛黄芩CHS基因正反义植物表达载体,并通过冻融法转化C58C1和LBA4404,为农杆菌介导的粘毛黄芩CHS基因对植物的遗传转化奠定基础。
OBJECTIVE: To obtain the sense and antisense plant expression vectors of chalcone synthase (CHS) gene of Viscidium scoparia, and further explore the mechanism of action of CHS on the pathway of biosynthesis of flavonoids by the transformation of Visco-skullcap root. Methods: The target fragment of CHS gene was amplified from total RNA of Radix Scutellariae by RT-PCR and cloned into PMD18-T vector for sequencing and bioinformatics analysis. Results: Sequencing proved correct sequence, at the same time deduced amino acid sequence of CHS and three-dimensional structure. The cloned fragment and pCAMBIA1304 + vector were double digested with BlgII and BstE II, and then the target fragment of CHS gene and the large framework fragment of pCAM-BIA1304 + vector were ligated to obtain the antisense and antisense plant expression vector of CHS. CONCLUSION: The positive and negative plant expression vectors of CHS gene were successfully constructed and transformed into C58C1 and LBA4404 by freeze-thaw method, laying a foundation for the genetic transformation of plant by Agrobacterium tumefaciens CHS gene.