Studies On The Catalytic Efficiency And Prokaryotic Expression Of Different Copies Of Licorice NCEDs

According to the "Shen Nong’s Materia Medica",China is the first country in the world to recognize and study licorice.Glycyrrhiza is a root and rootstock of Glycyrrhiza uralensis Fisch、Glycyrrhiza inflata Bat、or Glycyrrhiza glabra L,also known as sweetgrass.It is one of the four major medicinal materials collected and managed by the Chinese medical administra--tive department as a medicinal plant.It has the efficacy of supplementing spleen and qi,clearing heat and detoxification,removing phlegm,relieving cough,relieving pain,reconciling various drugs,and is widely used in clinical formulations.Pharmacological research mainly focuses on glycyrrhizic acid,glycyrrhetinic acid,total flavonoids,single flavonoids and polysaccharides,among which glycyrrhizic acid is the main medicinal component of licorice,and its content is one of the content determination items prescribed in the 2015 edition of the Chinese Pharmacopoeia of China.Research shows that in recent years,the cultivated licorice has a low content of glycyrrhizic acid,which does not meet the 2015 edition of the "Chinese Pharmacopoeia" standard and severely restricts the sustainable development and utilization of licorice resources.Therefore,it is very important to improve the quality of licorice by clarifying the relationship between the metabolism of each component in licorice and increasing the content of each medicinal component in licorice.Studies have shown that glycyrrhizic acid biosynthesis pathway is not isolated,it and other secondary metabolites such as abscisic acid(ABA),gibberellin(GA),cytokinin(6-BA),glycyrrhizin(Liquiritin),Liquiritigenin and other biosynthesis pathways connect to each other and form a network.Our previous study found that exogenous application of ABA at appropriate concentrations can increase the content of glycyrrhizic acid in licorice,and 9-Cis-Epoxycarotenoid Dioxygenase(NCED)gene it is one of the rate-limiting enzyme genes in the ABA synthesis pathway.Its expression level directly affects the synthesis of ABA.Therefore,it was concluded that the NCED gene is the key rate-limiting enzyme gene in the abscisic acid synthesis pathway,and the difference in the expression amount has a significant effect on the accumulation of ABA,and the difference in the content of externally applied ABA and the endogenous ABA plant can promote the accumulation of acid indicates that the difference in NCEDs gene expression can affect the activity of glycyrrhizic acid biosynthetic enzymes and eventually regulate the amount of glycyrrhizic acid synthesis.In this laboratory study,grey correlation analysis of SNPs of NCEDs of NCED1,NCED3,and NCED4 with ABA and glycyrrhizic acid were performed,respectively.Based on the results,it was preliminarily determined that the NCED1 gene mutation site had a better overall effect on the synthesis and accumulation of ABA during sampling than NCED3 and NCED4,but NCED3 and NCED4 genes also played a role in ABA synthesis and accumulation.Furthermore,the association coefficient of NCED1 gene 437 bp(G>A)and glycyrrhizic acid was found to be the largest among the three genes,followed by NCED3 gene 966 bp(G>A)and NCED4 gene 845 bp(A>G),that is to say,when selecting high glycyrrhizic acid germplasm,priority should be given to G-type at 437 bp for NCED1 gene,G-type at 966 bp for NCED3 gene and A-type at 966 bp for NCED4 gene.This experiment is based on this study to further verify the above conclusions and compare the differences in the content of abscisic acid and glycyrrhizic acid under the different catalytic functions of NCED1,NCED3,andNCED 4 that regulate the glycyrrhizic acid content.In this dissertation,transient overexpression methods were used to screen NCEDs for efficient copying based on the effects of three genes NCED1,NCED3,and NCED 4 on abscisic acid and glycyrrhizic acid content and different in vitro catalytic efficiencies,screening out highly efficient copies of NCEDs provides a theoretical basis for improving glycyrrhizic acid content and transgenic licorice research at the level of molecular regulation;At the same time,the NCEDs prokaryotic expression system was constructed to induce soluble protein and purify,which laid a foundation for the laboratory to further verify the function of NCEDs on the basis of prokaryotic expression.The main results achieved so far are as follows:(1)NCED1,NCED3 and NCED4 genes were cloned from Glycyrrhiza uralensis Glycyrrhizae plants,and were ligated with pMD-19T cloning vector.They were successfully transferred into E.coli DH5a competent cells and frozen at-80℃.(2)The root-specific universal expression vector was successfully constructed and the root-specific expression vectors carrying NCEDl,NCED3 and NCED4 genes were constructed on this basis.Construction of root-specific universal expression vector using T4 ligase linkage,The primers were designed according to the principle of protecting bases,and the modified synthetic TobRB7 promoter fragment was ligated between the two restriction sites of the vector pCAMBIA1305.1,Sa1Ⅰ and Bgl Ⅱ,and successfully transferred into E.coli DH5a competent cells.The PCR verification and sequencing validation results showed that the root-specific universal expression vector pCA1305.1-TobRB7 was successfully constructed.Based on the root-specific universal expression vector,a root-specific expression vector carrying NCED1,NCED3,and NCED4 genes was also constructed using the T4 ligase linkage method.The principle of primer design is the same as above,and the NCED1,NCED3,and NCED4 gene fragments are respectively connected to A successful root-specific universal expression vector pCA1305.1-TobRB7 has been constructed between the two Bgl II and Spe I sites and successfully transfected into E.coli DH5a competent cells.The results of PCR verification and sequencing validation showed that the expression vector pCAM-TobRB7-NCED1/3/4 was constructed successfully.(3)Successfully constructed licorice root-specific expression engineering bacteriaThe root-specific expression vectors pCA-TobRB7-NCED1,pCA-TobRB-NCED3 and pCA-TobRB7-NCED4 were transformed into Agrobacterium tumefaciens EHA105 competent cells by electroporation transformation method,respectively.Both the PCR verification and the sequencing verification results showed that the vector was successfully transferred,indicating that EHA-TobRB7-NCED1,EHA-TobRB7-NCED3 and EHA-TobRB7-NCED4 were successfully constructed.(4)Successful construction of Agrobacterium-mediated transient overexpression system of NCEDs geneThis article uses seed absorption method to construct Agrobacterium transient expression system.Method for transiently expressing foreign genes by using seeds to naturally absorb Agrobacterium resuspension,after infection with bacteria,GUS staining showed that blue spots appeared for the first time after 5 days of infection,blue spots disappeared after 14 days of infection,and no blue spots were seen by GUS staining of licorice seedlings not infected with Agrobacterium.Agrobacterium infection was successful.(5)Detection and differential analysis of the expression of glycyrrhizic acid key enzyme gene(3-AS and the content of abscisic acid and glycyrrhizic acid in transiently overexpressed licoriceIn this paper,the expression of key enzyme gene beta-AS in glycyrrhizic acid in the infected licorice seedlings infected with EHA-TobRB7-NCEDl,EHA-TobRB7-NCED3,and EHA-TobRB7-NCED4 was detected by fluorescent quantitative PCR.The results showed that there was a significant difference in the expression of β-AS gene between Glycyrrhiza uralensis infected by different engineering bacteria and the control group(P<0.05),the expression level of β-AS gene in the licorice group not treated with engineering bacteria was the lowest,and the expression level of β-AS gene after the engineering bacteria treatment group was significantly increased,and the degree of improvement was:EHA-TobRB7-NCED1>EHA-TobRB7-NCED3>EHA-TobRB7-NCED4.Enzyme-linked immunosorbent assay was used to determine the endogenous abscisic acid content in licorice from the infective group of licorice and non-engineered bacteria infected with EHA-TobRB7-NCEDl/NCED3/NCED4,the results showed that the content of endogenous abscisic acid in Glycyrrhiza uralensis was the lowest in the non-inoculated group,and the content of endogenous abscisic acid in Glycyrrhiza uralensis infected with the engineering strain EHA-TobRB7-NCEDl/NCED3/NCED4 was:EHA-TobRB7-NCED1>EHA-TobRB7-NCED3>EHA-TobRB7-NCED4.The contents of glycyrrhizic acid in Glycyrrhiza uralensis inoculated with licorice and non-inoculated liquid using EHA-TobRB7-NCED1/NCED3/NCED4 inoculation were detected by HPLC,the results showed that the content of glycyrrhizic acid in Glycyrrhiza uralensis was the lowest in the non-inoculated group.The content of glycyrrhizic acid in Glycyrrhiza uralensis infected with the engineering strain EHA-TobRB7-NCED1/NCED3/NCED4 was:EHA-TobRB7-NCED1>EHA-TobRB7-NCED3>EHA-TobRB7-NCED4.In summary,the above results explain that the G-type of the NCED1 gene sequence at 437 bp in the 3 genes was superior to the other two genes in the synthesis and accumulation of endogenous abscisic acid and glycyrrhizic acid during the sampling period.The NCED3 gene sequence was 966 bp at the G-type,ollowed by the NCED4 gene sequence.The type A was the weakest at 845bp,but NCED3 and NCED4 also had a certain role in the synthesis and accumulation of endogenous abscisic acid and glycyrrhizic acid.Therefore,the priority sites that should be considered when selecting glycyrrhizic acid for screening or conducting transgenic licorice research are G-type at 437 bp of NCED1 gene sequence,G-type at 966 bp of NCED3 gene sequence and A-type at 845 bp of NCED4 gene sequence.(6)NCED1,NCED4 soluble protein was successfully induced and successfully purified for the first time,NCED3 inclusion protein was induced and successfully purified.Based on the previous studies,this study optimized the induction conditions and induced the BL21(DE3)strain containing pET-30a(+)-NCED1/NCED3/NCED4 recombinant plasmid at 16℃ with IPTG overnight using the optimized induction conditions so that the target protein was expressed.According to the results of SDS-PAGE electrophoresis analysis,pET-30a(+)-NCED1 and pET-30a(+)-NCED4 expressed soluble proteins after induction,whereas pET-30a(+)-NCED3 was expressed as inclusion bodies after induction.The induced proteins were purified using different concentrations of imidazole,according to the analysis of the purified SDS-P AGE electrophoresis results,the protein expressed by pET-30a(+)--NCED1 and pET-30a(+)-NCED4 eluted at the imidazole concentration of 300 mM to produce a more pure single protein band,and then selected to elute at a concentration of 300 mM imidazole.The solution was ultrafiltered and the ultrafiltered protein was frozen.pET-3 0a(+)-NCED3 expressed as inclusion body after induction,after urea renaturation,eluting at the imidazole concentration of 100 mM yields a relatively pure single protein band,followed by ultrafiltration of the solution eluted at a concentration of 100 mM imidazole and freezing of the ultrafiltered protein.This will lay a theoretical foundation for further laboratory verification of NCEDs functions based on prokaryotic expressions in the future.