Effect Of Lactobacillus Plantarum On The Accumulation Of Calycosin In Astragalus Membrana Ceus Adventitious Roots

Astragalus membranaceus(Fisch.)Bunge is a genuine medicinal material in the northeast of China.Calycosin is one of its active components,which has multiple pharmacological activities.Adventitious roots culture is one of the ways to obtain the effective components of Astragalus membranaceus,but the content of calycosin in A.membranaceus adventitious roots(AMARs)is too low to be detected.A large number of studies have shown that the addition of elicitors can increase the content of plant secondary metabolites.Lactobacillus plantarum is a kind of probiotics.As an elicitor,L.plantarum can not only induce the accumulation of secondary metabolites,but also solve the biosafety.In this experiment,we used AMARs and L.plantarum as material and biological elicitors,respectively,to study the effects of the treatment duration and concentrations of L.plantarum on the accumulation of calycosin.Then the effect of L.plantarum culture filtrate and acid treatment on the accumulation of calycosin in AMARs were also studied.These results provide a theoretical basis for the production of calycosin in AMARs.The results showed that 2.0×109·mL-1 L.plantarum treated 96 h which induced the highest accumulation of calycosin in AMARs(46.6 μg·g-1).The conservative region cDNA of isoflavone conjugate-hydrolyzing β-Glucosidase(ICHG)gene of A.membranaceus was cloned.The qRT-PCR analysis showed that L.plantarum inhibited the gene expression levels of isoflavone synthetic pathway,but increased the expressions of ICHG.L.plantarum decreased the content of calycosin-7-O-β-D-glucoside(CG)while increasing the content of calycosin.Therefore,it is speculated that accumulation of calycosin induced by L.plantarum was from hydrolyzation of CG catalyzed by ICHG in AMARs.L.plantarum treatment decreased biomass,triggered the accumulation of nitric oxide,increased the EC of the culture medium,reduced the pH of the culture medium(pH=3.0);furthermore,calycosin was produced in the medium which was not produced in the control medium;meanwhile,L.plantarum did not have the ability to hydrolyze CG.All these results showed that L.plantarum had a stress effect on AMARs and AMARs produced calycosin as phytoalexin to resist L.plantarum.Similar to L.plantarum,L.plantarum culture filtrate can induce the accumulation of calycosin.Acid treatment(pH=3.0)did not affect the accumulation of calycosin,however,did induce the accumulation of CG.