The Effect Of Cytoplasmic Calcium Spurt To Salvia Miltiorrhiza Phenolic Acids Secondary Metabolites Induced By Salicylic Acid

This paper used Salvia miltiorrhiza Bunge as the materials and salicylic acid(SA) as the exogenous inducer. Using laser confocal microscope(CLSM) to detect cytoplasmic calcium ion, real time quantitative PCR to detect secondary metabolites synthesis. Real Time PCR to detect genes expression. High performance liquid chromatography(HPLC) to detect Salvia miltiorrhiza phenolic acids synthesis. To discuss SA induced cells Ca2 +signal’s effect in the synthesis of phenolic acids secondary metabolites. To provide a theoretical basis for the synthesis and regulation of secondary metabolites.In this paper we have made the following conclusions:(1) The method of preparating protoplasts of salvia miltiorrhiza suspension culture cells has been getted. Enzymatic hydrolysis conditions are cellulase R-10 1.5%, pectinase Y-230.3%, Macerozyme R-10 0.5%. enzymolysis 12 hours. Appropriate mannitol concentration is0.4 mol/L. 40 r/min 12 h, 600 r/min 5 min, and the protoplasts yield was 1.1×106 cells/g FW,the vitality was more than 95% by using fluorescein diacetate staining method.(2) SA treatment can induce salvia miltiorrhiza cell cytoplasm Ca2+burst. Cytoplasmic Ca2+ spurt comes mainly from extracellular calcium library.cBy using CLSM it has been observed that SA treatment can rapidly cause salvia miltiorrhiza protoplasts cytoplasmic Ca2+burst. In 0 ~ 90 s cytoplasmic Ca2+concentration rising rapidly, and reaching the peak at12 min. Intracellular calcium reservoir inhibitor(2-APB) and plasma membrane Ca2+ channel inhibitor(LaCl3) pretreatment both can inhibit the SA treatment caused cytoplasmic Ca2+burst, but the inhibition effect of La Cl3 is more remarkable. These results indicate that the extracellular Ca2+ induced by SA is the main source of the cytoplasm Ca2+ burst.(3) SA induced cytosolic calcium signaling promoted the biosynthesis of SalB by regulating the expression of related enzymes. The expression of RAS, TAT and PAL genes were up-regulated obviously in the secondary metabolites biosynthesis of Salvia miltiorrhiza.La Cl3 and 2-APB pretreatment both can inhibit the expression of SA and the increase of phenolic acids secondary metabolites contents, and the effect of La Cl3 is more obviously. The expression of the gene and the contents of phenolic acids was up-regulated by exogenous addition of excess Ca2+ and Ca2+ carrier. These results indicate that the Ca2+ signal of thecytoplasm plays an important role in the process of the secondary metabolite biosynthesis mediated by SA.The above research results revealed the Ca2 + signaling and its regulation mechanism in SA induced promotion of secondary metabolites synthesis and the signal transduction pathways, and laid a foundation for regulating secondary metabolites synthesis by using signal transduction pathway.