Study On The Heating Mechanism Of Ginger Extract In Yeast Model

Ginger,which has a pungent taste,is slightly warm,and can be used to prevent cold.It has the biological effects of anti-cancer,anti-oxidation and anti-inflammatory.However,its heating mechanism is still unclear.The ginger oleoresin used in this thesis is a dark brown viscous liquid extracted from ginger and composed of volatile oils and gingerols,which are the main components of ginger.In this thesis,we attempt to use Saccharomyces Cerevisiae as a model to explore the heating mechanism of ginger oleoresin.In this thesis,Saccharomyces cerevisiae BY4741 and its mutant strains were used as models to study the phenotype and genotype based on the classical thermal signaling pathways Hog1 and Pkc1 in Saccharomyces cerevisiae.The phenotype is mainly indicated to the growth curve analysis and the gradient dilution analysis.The growth curve detects the growth of yeast cells by the full-automatic growth curve tester,and the gradient drip test can further observe the effect of ginger oleoresin from the growth state of the cells.The genotype was mainly discussed from the level of mRNA to protein.In this thesis,the relative expression of heat shock protein induced by ginger oleoresin was tested by Real-time PCR.Phosphorylation of kinases and transcription factors was observed at the protein level using Western-blotting experimental techniques.The research in this thesis indicates that ginger oleoresin can affect the growth rate of wild type strain BY4741 WT,Sho1?,Pbs2?,Hog1?,Mpk1?,Mid2?,Rom2?,Bck1? strains,and the time to reach logarithmic growth phase is significantly prolonged.It is preliminarily judged that ginger oleoresin may be simulated with high temperature in yeast in response to the stimulations and there are at least two signal pathways involved.The expression of heat shock protein Hsp82 was detected by Real-time PCR.The results showed that the effect of Hog1 signaling pathway was more significant when ginger oleoresin was applied to yeast cells.When ginger oleoresin stimulated wild type strain WT,the expression of heat shock protein HSP82 was up-regulated.But the increment of Hsp82 was reduced in the Hog1? and Po4?(Hsf1)deletion strain,and there was a significant decrease compared with the wild type.However,the key strains of the Pkc1 signaling pathway did not show significance,so the stimulation of the Pkc1 pathway was excluded.The kinase Hog1 and the transcription factor Hsf1,which are mainly targeting the Hog1 signaling pathway,were subsequently studied.The results of transcription factor nuclear assay and Western-Blotting experiment suggested that when ginger oleoresin apply on yeast cells,it will transmit signals to the kinase Hog1,after receiving the signal,Hog1 will rapidly phosphorylate,and Hog1-P will enter the nucleus.Then the transcription factor Hsf1 enters the nucleus and is transcribed,the Hsf1 transcription factor is also phosphorylated upon activation,regulating the expression of downstream genes.We can observe a significant up-regulation of the Hsf1-GFP protein band and confirm the subsequent addition of phosphatase and phosphatase inhibitors,indicating that the thermogenic mechanism of ginger oleoresin is related to the Hog1 signaling pathway,but due to the absence of Hog1 is not the only regulatory kinase,and it is speculated that there will be synergistic regulation of Hsf1 by other kinases.Therefore,the heating of ginger oleoresin may be a synergistic effect of Hog1 signaling pathway and other potential stress mechanisms.In general,this thesis also reveal the heating mechanism of ginger oleoresin to a certain extent,and provide a basis for the thermal research of other natural products,and further facilitate the fermentation industry.