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药用植物遗传转化体系的建立对其功能基因研究具有重要意义,该研究在已有苦豆子再生体系的基础上,对农杆菌菌液浓度、农杆菌侵染时间、农杆菌与苦豆子愈伤组织的共培养时间、愈伤组织预培养时间、乙酰丁香酮添加方式和乙酰丁香酮浓度6个遗传转化因子进行优化,结果表明在预培养15 d的苦豆子愈伤组织中农杆菌菌液浓度A600为0.9、侵染时间15min、农杆菌与愈伤组织共培养48 h、并在侵染液中添加AS 200μmol·L~(-1)时,GUS瞬时转化效率高达83.33%。以苦豆子基因组DNA为模板克隆获得Sa LDC上游1 260 bp的启动子序列(Gen Bank登录号KY038928),将不同长度(310,594,765,924,1 260 bp)的启动子缺失片段分别与GUS报告基因融合,构建的植物表达载体经农杆菌介导转化苦豆子愈伤组织,GUS瞬时表达结果显示,5个不同长度的Sa LDC启动子片段均可驱动GUS在苦豆子愈伤组织中的表达,表明克隆所得的Sa LDC启动子具有启动活性,以310 bp的片段启动活性最强,为进一步分析该启动子功能奠定了基础。
The establishment of medicinal plant genetic transformation system is of great significance to the study of its functional gene. Based on the existing regeneration system of Sophora alopecuroides, the study on the Agrobacterium tumefaciens concentration, Agrobacterium tumefaciens infection time, Agrobacterium tumefaciens and Sophora alopecuroides The results showed that Agrobacterium tumefaciens concentration of A600 in callus of Sophora alopecuroides cultured for 15 days was significantly higher than that of the control Was 0.9 and the infection time was 15 min. The Agrobacterium tumefaciens co-cultured with callus for 48 h, and the GUS transient transformation efficiency was as high as 83.33% when AS 200 μmol·L -1 was added to the inoculum. The 1 260 bp upstream of Sa LDC (Gen Bank accession number KY038928) was cloned from the genomic DNA of Sophora alopecuroides. The deletion fragments of promoters with different length (310,594,765,924,1 260 bp) were fused with GUS reporter gene respectively to construct Of Agrobacterium-mediated transformation of Sophora alopecurophytica callus, transient expression of GUS results showed that five different lengths of the Sa LDC promoter fragment can drive GUS expression in the callus of Sophora alopecuroides, indicating that the cloned The promoter of Sa LDC promoter has the highest promoter activity with a 310 bp fragment, which lays the foundation for further analysis of the function of this promoter.