Cloning Of GuGDHs And GuCPR Genes In Glycyrrhizic Acid Biosynthesis Pathway And Analysis Of GuGDHs Activity

Glycyrrhiza uralensis Fisch.has the effects of replenishing spleen,clearing away heat and detoxifying,relieving cough,relieving pain and relieving pain.Glycyrrhizic acid is the main active ingredient of Glycyrrhiza uralensis Fisch.The content of glycyrrhizic acid is the main indicator for measuring the quality of medicinal herbs in Chinese Pharmacopoeia.The medicinal value and industrial value of glycyrrhizic acid are remarkable,not only used in clinical treatment,but also widely used in cosmetics,food,health care products and other industries.Currently,the cultivation of licorice alone cannot meet its huge market demand.At the same time,the licor:ice planting has a long cycle and the quality is different.The tran sitional mining of Glycyrrhiza uralensis Fisch.causes serious problems such as land desertification,soil infertility and ecological environment damage.Therefore,adopting emerging science and technology The acquisition of glycyrrhizic acid has become a hot issue in the research of Glycyrrhiza uralensis Fisch.resources.In recent years,with the rapid development of synthetic biology technology,the research on the biosynthesis pathway of glycyrrhizic acid has become more and more mature.Therefore,the use of synthetic biotechnology to synthesize glycyrrhizic acid is expected to alleviate the shortage of glycyrrhizic acid market and improve the germplasm resources of licorice.An important method.Currently,most of the key genes in the glycyrrhizic acid biosynthesis pathway have been successfully characterized,and the biosynthesis pathway of glycyrrhizic acid has been basically opened.Scientists have synthesized glycyrrhetinic acid precursor,glycyrrhetinic acid,in yeast.However,UDP-glucose dehydrogenase(GuGDHs)and cytochrome P450 reductase(GuCPR)in the midstream and downstream pathways of glycyrrhizic acid have not been successfully resolved.Glycyrrhizin glycosyltransferase needs to be catalyzed by UDP-glucose dehydrogenase(GuGDHs)to catalyze the production of glycyrrhizic acid as the final product glycyrrhizic acid;glycyrrhizic acid cytochrome P450 reductase(GuCPR)is the catalytic process of cytochrome P450 Providing electrons is an important member of the cytochrome P450 superfamily enzyme.Therefore,analysis of glycyrrhizic acid UDP-glucose dehydrogenase(GuGDHs)and glycyrrhizic acid cytochrome P450 reductase(GuCPR)is essential for the biosynthesis of glycyrrhizic acid.Therefore,the research content and main research results of this paper are as follows:1.Through the transcriptome database of the previous transcriptome of this group and the homologous genes reported on NCBI,three candidate glycyrrhizic acid UDP-glucose dehydrogenases(GuGDHs)were selected and named as GuGDH1,GuGDH2 and GuGDH3;A full-length coding sequence of glycyrrhizic acid cytochrome P450 reductase(named GuCPR),using cDNA as a template,PCR-based(Polymerase Chain Reaction),specific primer amplification,cloned GuGDHl,GuGDH2,GuGDH3 and GuCPR complete coding region(CDS),three bands of about 1000 bp and one 2,000bp were obtained,and the recombinant plasmids of GuGDH1,GuGDH2,GuGDH3 and GuCPR were obtained by ligating pEASY-Blunt vector.Based on protein structure prediction and phylogenetic tree analysis,it was shown that GuGDH1,GuGDH2 and GuGDH3 belong to the UDP-glucose dehydrogenase superfamily,and GuCPR belongs to the cytochrome P450 reductase superfamily.In addition,the basic characteristics of GuGDHs and GuCPR were analyzed using relevant bioinformatics analysis methods.2.The prokaryotic expression vector of pET-30a(+)of GuGDH1,GuGDH2 and GuGDH3 was constructed by homologous recombination method and heterologously expressed in E.doli BL21(DE3).The expression of GuGDH1,GuGDH2,and GuGDH3 was obtained by inducing expression by IPTG(Isopropyl P-D-Thiogalactoside).3.Enzymatic reaction catalysis experiments were carried out to detect the increase of absorbance of NADPH at 340nm to determine the activity of glycyrrhizic acid UDP-glucose dehydrogenase(GuGDHs),which proved that GuGDH1 and GuGDH2 were active,while GuGDH3 was not active.The research results in this paper can lay the foundation for the further analysis of glycyrrhizic acid biosynthesis pathway.