Establishment Of Genetic Transformation System And Construction Of Subtractive CDNA Library Under Nacl Stress Of Cultured Cells Of Glycyrrhiza Inflata

Licorice was one of the most commonly used herbs in traditional Chinese Medicine.Limited by many objective conditions, the main medicinal secondary metabolites contentedin licorice by artificial cultivation was very low, which affected the medicinal value oflicorice. With the development of biotechnology, to regulate the secondary metabolismpathway in licorice and improve its medicinal value through biotechnology method becamefeasible.The callus of Glycyrrhiza inflata screened in our laboratory, have the features of rapidgrowth, genetic stability, and high producing of secondary metabolites, such as licoriceflavonoids, licorice polysaccharides. The cultured cells of G. inflata were suitable for study ofsecondary metabolism in licorice.In this study, the Agrobacterium tumefaciens-mediated genetic transformation system ofG. inflata callus was established. Two of A. tumefaciens strains, EHA105and LBA4404, bothcontaining a binary vector pBI121-gfp carrying neomycin phosphotransferase II (NPT-II),green fluorescent protein (gfp) genes were used. The transformation efficiency under theconditions of different period pre-cultured callus and different infecting time with A.tumefaciens were analyzed. The results showed that optimum genetic transformationcondition was using G. inflata callus subcultured for7d as target tissue, infection withEHA105-gfp for15min, co-culture on medium for3d, transformed calli were obtained aftercultured on selection medium containing100mg/L kanamycin for45d. Transformation wasconfirmed by a fluorescence detection and PCR amplification of the gfp gene. The maximumtransformation rate was97.14%.A subtractive cDNA library was constructed by suppression subtractive hybridizationmethod using cDNA of G.inflata callus as driver and G.inflata callus treated under salt stressas tester.By compared with primer1in the first PCR and the nested primers in second PCR,983positive clones were obtained through the blue white screening method, and therecombination rate of the cDNA library was85.7%.60positive clones were selected to analyze the sequence. And the results of EST sequence analysis showed that the clones withputative functions and the clones with unknown functions were comprised of the percentageof75%and25%, respectively. These genes are involved in ubiquitin, mRNA, phloemproteins.And it was highly homologous with white spruce, wheat, barley and maize. Thefunctions in the plant involved in the transcriptional regulation and protein synthesis whichwas possibly of G.inflata callus under salt stress related genes.