Effects Of Non-biological Factors On The Expression Of Key Enzyme Genes Involved In Ginsenosides Biosynthesis Pathway

Panax ginseng,belonging to Araliaceae,is a precious traditional Chinese Medicine.Ginsenosides,the main secondary metabolites of ginseng,have a wide application prospect and considerable economic value.With a variety of pharmacological effects,such as improving immunity and fatigue,anti-aging and anti-tumor,ginsenosides can be used in industries like health care,cosmetic,food.However,because of the lack of wild resources of ginseng,the long period of cultivation,the vulnerability to diseases,the high requirement of planting technology,and especially the less content of ginsenosides,the development and utilization of ginseng are under certain restrictions,which makes the social needs cannot be properly satisfied.Studies have shown that external stimulating factors,such as non-biological stress and exogenous hormone treatment,can activate intracellular signal transduction pathway,transmit the signal to the stress responsive transcription factor,regulate the expression of a series of key enzyme genes and affect the synthesis of secondary metabolites,so that the plant can finally have its stress tolerance.In this study,the expressions of three ginseng transcription factor genes(WRKY,ERF,MYB)and seven ginsenoside biosynthesis key enzyme genes(HMGR,DS,D12 H,P6H,UGT-D-3OH-1,UGT-D-3OH-2 and UGT-D-20OH)under four different treatment conditions(salt stress,low temperature stress,exogenous MeJA treatment and exogenous ABA treatment)are discussed.Morever,the relationships between them have also been analyzed.The main contents and results of this study are shown as follows:(1)The CDS sequence of the ginseng transcription factor WRKY1 gene was successfully cloned and named Pg WRKY1.The sequencing results showed that the sequence length of PgWRKY1 gene was 1077 bp,which could encode 358 amino acids.In addition,the sequence alignment results showed that the similarity of Pg WRKY1 and Pq WRKY1 was 99.16% in nucleotide sequence while 98.32% in amino acid sequence.After submitting the relevant information to GenBank,the search number can be obtained as KU836935.(2)The CDS sequence of the ginseng transcription factor ERF1 gene was successfully cloned and named PgERF1.The sequencing results showed that the sequence length of PgERF1 gene was 936 bp,which could encode 311 amino acids.Furthermore,the sequence alignment results showed that the similarity of PgERF1 and PqERF1 was 98.29% in nucleotide sequence while 97.75% in amino acid sequence.After submitting the relevant information to GenBank,the search number can be obtained as KU935742.(3)The core fragment of the PgHMGR,PgDS,PgD12 H,PgP6H,PgUGT-D-3OH-1,PgUGT-D-3OH-2,PgUGT-D-20 OH,PgMYB,PgWRKY1 and PgERF1 were cloned.The results of electrophoresis showed that the PCR reaction procedure was suitable and the accuracy and specificity of the primers were better.(4)Under both low temperature and MeJA conditions,the expressions of seven key enzyme genes were increased.Under salt stress,the expressions of PgHMGR,PgP6 H and PgUGT-D-20 OH were increased,the expression of PgD12 H was not obvious,and the expressions of PgDS,PgUGT-D-3OH-1 and Pg UGT-D-3OH-2 were decreased.Under ABA treatment,the expression of seven key enzyme genes was decreased.(5)Under low temperature stress,the expressions of transcription factor genes PgMYB and PgERF1 were increased,and the expression of PgWRKY1 was not obvious.Under salt stress,the expressions of three transcription factor genes were increased.Under MeJA treatment,the expressions of PgMYB and PgWRKY1 were increased,and the expression of PgERF1 was decreased.Under ABA treatment,the expressions of PgWRKY1 and PgERF1 were increased,while the expression of PgMYB was decreased.(6)The total saponins content of ginseng hair root were measured by Vanillin colorimetric method,which showed that compared with the control group(untreated),the total saponins content of ginseng hair root was increased by about 50%,17% and 8% respectively under MeJA treatment,low temperature stress and salt stress,while the total saponins content under ABA was reduced by 9% approximately.(7)The relationship between transcription factors and key enzymes: there may be a positive correlation between Pg MYB and PgD12 H gene,while negative correlation between PgERF1 and PgUGT-D-3OH-1,PgUGT-D-3OH-2.