Mechanism Of Enzymatic Browning In Cell Culture Of Glycyrrhiza Uralensis Fisch

The dried roots and rhizomes of Glycyrrhiza uralensis Fisch.are commonly used Chinese medicinal materials.They have the effects of Tonifying the spleen,clearing away heat and detoxification,eliminating phlegm and relieving cough.With the increase of clinical dosage and the expansion of development and utilization scope,the demand for Glycyrrhiza has increased sharply.The production of effective components of medicinal plants by large-scale culture of plant cells is due to its rapid growth rate,controllable quality and independent growth environment are considered to be one of the most potential means of biotechnology industrialization,but there are still a series of problems in the process of industrialization,including cell browning,which is the most common and happen through the whole culture process.Browning seriously affects cell growth,resulting in the accumulation of cell biomass and the decrease of secondary metabolite production.At present,there is no clear understanding of the mechanism of browning in suspension system,and no effective measures are put forward.In this study,Glycyrrhiza uralensis cells were taken as the research object,and the effects of specific environment on cell browning were analyzed by changing osmotic pressure,dissolved oxygen,fluid shear force and other factors after solid-liquid transformation,and the main influencing factors were determined.At the same time,metabolomics and transcriptome analysis were carried out on cells with different browning degrees to determine the main substrates and key genes involved in enzymatic Browning,and clone related genes,so as to preliminarily explore the mechanism of enzymatic Browning in plant cell culture.The main results are as follows:(1)By adjusting osmotic pressure,fluid shear force,dissolved oxygen and other conditions of cell suspension system of Glycyrrhiza uralensis,the effect on cell browning(intracellular total phenol,PPO activity,browning degree)was detected.The results show that these environmental factors have certain influence on browning,high osmotic pressure,high fluid shear force and dissolved oxygen all increase the browning degree.The occurrence of cell browning can be controlled by regulating the concentration of sucrose in the medium and the rotation speed of the shaking table in the initial stage of suspension culture.(2)The metabolomics of cells with different browning degrees were analyzed by LC-MS~n technology.The results showed that with the increase of cell browning degree,the content of phenols as enzymatic browning substrate changed significantly,mainly flavonoids and isoflavones.These two compounds may participate in the occurrence of enzymatic browning as main substrates.The synthesis of flavonoids and isoflavones in plant cells comes from phenylpropane pathway,and the upstream is shikimic acid pathway and glycolysis pathway respectively.The changes of their biosynthesis must be regulated by these three pathways.(3)RNA-seq technology was used to sequence the transcriptome of cells with different browning degrees.The results showed that 75 differentially expressed genes were in phenylpropane synthesis pathway,shikimic acid synthesis pathway and glycolysis pathway,of which 23 were directly involved in the branch pathway of flavonoids and isoflavones synthesis.Through q PCR verification,the expression level of eight genes in browning cells is higher than that in normal cells,which is consistent with the results of transcriptome sequencing such as Gl PK,Gl PAL,Gl24CL,Gl1PDT,Gl3CHI,Gl C4H,Gl2F3’H,Gl2CCR.They are key genes involved in the occurrence of cell browning.At the same time,11 MYB transcription factors related to cell browning were screened.(4)The key genes Gl PK,Gl3CHI,Gl2CCR,Gl MYB1 were cloned and analyzed by bioinformatics,which provided a basis for further controlling the occurrence of browning in plant cell suspension culture by regulating the expression of these genes.