A Preliminary Study On The Molecular Mechanism Of Moderate Drought Stress Regulation On The Secondary Metabolism In Glycyrrhiza Uralensis

Glycyrrhiza uralensis is a herbaceous of perennial legume, the roots and rhizomes ofwhich named licorice are used as medicine. It was effective medicament for spleen qi,detoxification, expectorant cough, urgency and pain relief, and can reconcile the efficacyof various drugs. With the growing demand of licorice, it has led to over-exploitation ofG. Uralensis resources. Protection of wild G. uralensis resources and improve the qualityof cultivated licorice is a key factor in solving this problem.G. uralensis is mainly distributed in the northwest, north and northeast of China,suitable for growing in arid and semi-arid environments. Soil water conditions can directlyaffect the yield and quality of licorice. The research shows that moderate drought stress(soil relative water content of40%~45%) can promote accumulation of some activeingredients, such as liquiritin and glycyrrhizic acid, and therefore improve the quality ofthe licorice. In this paper, based on the6months licorice experiment material afterprocessing with moderate drought stress, we researched the molecular mechanism ofdrought stress regulation of licorice secondary metabolism by transcriptome sequencing.It can improve the active ingredients content of cultivated licorice, and lay the foundationfor sustainable use of licorice resources.Weighing replenishment method was used to control the degree of drought stress.Content of liquiritin and glycyrrhizic acid of the two groups was determined by HPLC. Weuse the transcriptome sequencing technology to obtain genetic information in response todrought stress, then key genes that regulating the biosynthesis of the active ingredients oflicorice under moderate drought stress was screened out by functional annotation andclassification. Based on the sequences obtained by transcriptome sequencing, taxadiene 5-α-hydroxylase and squalene synthase was selected for the fpllowing study.Semi-quantitative RT-PCR technique was applied to analyze the regulation of geneexpression under drought stress. We have utilized the bioinformatics methods to predictephysical and chemical properties of licorice Taxadiene5-α hydroxylase (GuT5H), as wellas structure and function of the cDNA sequence that encoded this protein, layingfoundations for further study of the gene functions and resolving molecular mechanismsof promotion of active ingredients accumulation under moderate drought stress.The research results are as follows:1. Contents of liquiritin and glycyrrhizic acid in the moderate drought stress andcontrol groups determined by HPLC shows that active ingredients content of moderatedrought stress group was significantly higher than that of control group.2. Transcriptome sequencing of the two groups was accomplished. After stitching weget Unigenes and All-unigenes which lengths are between300nt and3000nt, the averagelength being1400nt. The differentially expressed genes was screened out by RPKMmethod. Moderate drought stress have siginificant regulation effect on the pathway genesof secondary metabolism, stress response, signal transduction, and so on. Show thatdrought stress regulation the growth and development, accumulation of effectivecomponents in G. uralensis with multimodal pathways. It involves the interaction of thestress signal and phytohormone regulation factor signaltransduction.3. Expression patterns of taxadiene5-α hydroxylase and squalene synthase bysemi-quantitative RT-PCR analysis shows that upregulated expression of the two proteinsoccurs in moderate drought stressed licorice.4. The bioinformatics results shows that cDNA of GuT5H was1602bp, with an openreading frame of1428bp encoding a protein of475amino acid residues. GuT5H hasclosest evolutionary relationship with T5H of soybean, alfalfa and chickpea, whichencodes taxadiene5-α hydroxylase and belongs to the cytochrome P450family.