Correlation Of Glycyrrhizin ?-AS, ARPI And UGE Genes With Glycyrrhizic Acid Biosynthesis

Licorice is one of the most commonly-used Chinese herbs in China.It possesses various pharmacological activities and has a huge market demand.The Chinese Pharmacopoeia stipulates that glycyrrhizic acid(GA)is one of the marker components in licorice and has important medicinal value.Functional genes involved in the secondary metabolic pathway play an important regulatory role in the biosynthesis of GA in licorice.In our previous transcriptome analysis of Glycyrrhiza glabra,it was found that ?-amyrin synthase(?-AS),Auxin-responsive protein IAA(ARPI)and UDP-Galactose/Glucose-4-epimerase(UGE)genes were closely related to the GA accumulation.Based on the previous transcriptome sequencing results,this paper uses reverse genetics strategies to reveal the functions of these three genes.?-AS,ARPI,UGE gene silencing and overexpression vectors were constructed and transformed into ATCC15834 by electroporation,and which were used to induce licorice hairy root lines overexpressing and silencing ?-AS,ARPI and UGE genes.The content of GA in each hairy root line was assayed using UPLC.The functions of?-AS,ARPI and UGE genes on GA biosynthesis were clarified.The following results were obtained in this paper:(1)Induction and construction of licorice hairy root lines silencing ?-AS,ARPI and UGE genes:The sgRNA sequences of ?-AS,ARPI and UGE genes were designed using on-line tool(https://zlab.bio/guide-design-resources),respectively.The sgRNA sequences were denatured,annealed and then ligated to the linearized pHSE401 vector.The silencing vectors were transformed into Agrobacterium rhizogenes ATCC 15834 by electroporation.Licorice hairy root lines silencing ?-AS,ARPI and UGE genes were induced by infecting licorice sterile seedlings with recombinant A.rhizogenes strains.?-AS gene silencing licorice hairy roots grew normally,while ARPI and UGE gene silencing hairy roots died after 4 weeks of culture.It was speculated that ARPI and UGE were indispensable genes for the growth of licorice hairy roots.After verification by PCR and sequencing,7 ?-AS gene silencing licorice hairy root lines were obtained,and the editing efficiency was 46.7%.(2)Induction and construction of licorice hairy root lines overexpressing ARPI and UGE genes:The binary expression vectors pCA-ARPI and pCA-UGE overexpressing ARPI and UGE genes were transformed into A.rhizogenes ATCC15834 by electroporation.The recombinant A.rhizogenes ATCC15834 strains were used to infect licorice sterile seedlings to induce hairy root lines which were identified and screened by PCR and sequencing.Since the related research about licorice hairy roots overexpressing ?-AS gene has been carried out in our previous studies,it was not repeated in this paper.(3)Induction and construction of wild type and negative control licorice hairy root lines:Vectors pHSE401 and pCMBIA1305.1 were transformed into A.rhizogenes ATCC15834 by electroporation.A.rhizogenes ATCC15834 and recombinant ATCC15834-pHSE401 and ATCC15834-pCMBIA were used to infect licorice sterile seedlings to induce hairy roots,which were identified and screened by PCR and sequencing.(4)Determination and analysis of GA content in licorice hairy root samples:The GA content in 23 licorice hairy root samples,including wild type group(1 sample),negative control group(2 sample),ARPI overexpressing group(5 sample),UGE overexpressing group(8 sample)and ?-AS silencing group(7 sample)was assayed by UPLC.Statistical analysis showed that GA was not present in ?-AS silencing group.The GA content in ARPI overexpressing group showed a large difference.In sample ARPI+-3 the GA content was significantly higher than that in the wild type sample,while in the other four samples the GA content was significantly lower than that in the negative control sample.The GA content in all the 8 samples in UGE overexpressing group was higher than that in the wild type sample and negative con trol sample,and UGE+-1 was the highest.The GA content data was also statistically managed and compared among groups.It was found that the GA content in ARPI overexpressing group was significantly lower than that in negative control group,while the GA content in UGE overexpressing group was significantly higher than that in both wild type and negative control groups.(5)Correlation analysis between ?-AS,ARPI,UGE genes and GA biosynthesis:In this study,GA was not present in ?-AS silencing group,which demonstrates that silencing ?-AS gene causes the failure of triterpene skeleton formation and subsequently blocks the biosynthesis of ?-aromatic alcohol and its downstream products,confirming the crucial role of ?-AS gene in the downstream of GA biosynthetic pathway.ARPI silencing caused the death of licorice hairy roots,while ARPI overexpression did not increase the GA content in licorice hairy roots,which demonstrates that ARPI gene is necessary for the growth and development of licorice hairy roots,but it has no direct effect on the GA content.We consider the possible reason is as follows:ARPI contains a transcription inhibitory domain,thus overexpression of ARPI cannot effectively increase the auxin content.Therefore,both the utilization of carbon sources and GA biosynthesis are not increased in licorice hairy roots.UGE silencing caused the death of licorice hairy roots,and UGE overexpression significantly increased the GA content in licorice hairy roots,which demonstrates that UGE gene is necessary for the growth and development of licorice hairy roots,and it also plays an essential role in the biosynthesis of GA.UGE catalyzes the mutual conversion between UDP-galactose and UDP-glucose,which is the rate-limiting step in the biosynthesis of polysaccharides.Overexpression of UGE effectively improves the biosynthesis of primary metabolites,promotes the growth of hairy root cell walls,and increases the content of substrates involved in the secondary metabolism,which finally leads to the significant increase of GA content in licorice hairy roots.