Establishment of genetic transformation system for Sophra alopecuroides and deletion analysis of SaLDC promoter / 中国中药杂志

ABSTRACT

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-1AS. 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.