苦豆子遗传转化体系建立及SaLDC启动子缺失分析

Establishment of genetic transformation system for Sophra alopecuroides and deletion analysis of SaLDC promoter

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。

Establishing the genetic transformation system of medicinal plant is important to study their functional genes. Based on the established regeneration system of Sophra alopecuroides, 6 factors of genetic transformation were optimized, that was the concentration of Agrobacterium tumefaciens, the infection time, the co-cultivation time of agrobacterium tumefaciensand S.alopecuroides callus, the preculture time of S.alopecuroides callus, the adding method ofacetosyringone （AS） and the concentration of AS, respectively. The results showed that a maximum genetic transformation efficiency of 83.33% was achieved with 15d-precultured of S.alopecuroides callus, which was infected by A600=0.9 A. tumefaciens for 15 minutes and then co-cultivated for 48 hours with 200 μmol·L^-1 AS. The promoter sequence （1 260 bp） of upstream SaLDC was cloned from S.alopecuroides genomic DNA （gene bank accession number：KY038928）. The deletion fragment of SaLDC promoter with different length （310,594,765,924,1 260 bp） were ligated with the GUS reporter gene to form five plant expression vectors named P310,P594,P765,P924,P1260, which were then transferred into S.alopecuroides callus. The GUS transient expression showed that all 5 different deletion fragment of SaLDC promoter can drive the GUS gene expression in S. alopecuroides callus. The SaLDC promoter we cloned has high promoter activity, and they may facilitate its function analysis in the future.