The Mechanism Of ABA-responsive GubZIP Transcription Factors In Regulating The Biosynthesis Of Active Components In Glycyrrhiza Uralensis Fisch.

Licorice is a well-known medicinal material in China,which is valued for its ability to tonify spleen and qi,clear up heat and toxin,and reconcile various medicines.Its primary source is Glycyrrhiza uralensis Fisch.,and its main components are flavonoids and triterpenoids.However,the quality of cultivated licorice is often unstable and does not meet the standard of the Pharmacopoeia of the People’s Republic of China.Previous studies have shown that treatment with an appropriate concentration of abscisic acid(ABA)can significantly increase the content of active ingredients in G.uralensis.Nonetheless,the mechanism by which ABA regulates the synthesis of active ingredients remains unknown.To investigate this,our research group used Illumina high-throughput sequencing technology to perform transcriptome sequencing on G.uralensis treated with different concentrations of ABA.Through this approach,transcription factor genes(GubZIP1,GubZIP5,GubZIP33,and GubZIP56)responding to ABA in plants of G.uralensis were screened and cloned.In this study,the hairy root system of G.uralensis was used to screen GubZIPs in response to ABA,and the spatiotemporal expression characteristics of both the GubZIPs and key enzyme genes involved in the biosynthesis of active components(KEG-BIOs)of G.uralensis were analyzed.The promoters of the KEG-BIOs were cloned based on the genome of G.uralensis,and their cis-acting elements were analyzed.The specific binding sites of both the GubZIPs and KEG-BIOs in G.uralensis were verified through yeast onehybrid assay(Y1H).Additionally,a prokaryotic expression system for the GubZIPs was successfully constructed and the soluble target proteins were obtained.The main results of this study are as follows:1.GubZIP1,GubZIP5,GubZIP33,and GubZIP56 were significantly responsive to ABA in the hairy roots of G,uralensis.Both GubZIP1 and GubZIP5 were highly expressed in the root of licorice.The expressions of GubZIP1,GubZIP5,and GubZIP56 genes increased with the growth of licorice.The response of GubZIPs to ABA was studied using the hairy root system of G.uralensis,which revealed that GubZIP1,GubZIP5,GubZIP33,and GubZIP56 exhibited significant responses to exogenous ABA treatment.The four GubZIPs showed the most significant response to ABA when treated with 50 mg·L-1 ABA for 3 h.The spatiotemporal expression characteristics of GubZIPs in G.uralensis were different.Real-time qPCR results indicated that GubZIP1 and GubZIP5 were highly expressed in the root of G.uralensis.These findings were consistent with the expression patterns of KEG-BIOs,such as CHS and CHI in the liquiritin synthesis pathway,as well as HMGR,SQS,β-AS,CYP88D6,CYP72A154,GuCSyGT,and UGT73P12 in the glycyrrhizic acid synthesis pathway.It was suggested that GubZIP1 and GubZIP5 might be key regulatory factors in the biosynthesis pathway of active components in G.uralensis.The expression levels of GubZIP1,GubZIP5,and GubZIP56 were consistent with the expression of CHI,HMGR,SQS,and UGT73P12,which increased with the growth of licorice.The expression level of GubZIP33 was the highest in two-year licorice,but there was no significant difference between one-year and three-year licorice,which was consistent with the expression pattern of β-AS,CYP88D6,CYP72A154,and GuCSyGT.2.The promoters of CHS,CHI,SQS,β-AS,and GuCSyGT contain cis-acting elements that can bind to GubZIPs.The promoter sequences of CHS,CHI,HMGR,SQS,β-AS,CYP88D6,CYP72A154,GuCSyGT,and UGT73P12 were extracted from G.uralensis genome by TBtools software.The results of cis-acting elements analysis showed that the promoters of nine key enzyme genes in G.uralensis contained a large number of cis-acting elements responding to hormones or external stress.The promoters of CHI,CHS,β-AS,SQS,GuCSyGT,and UGT73P12 were cloned successfully.The results of cis-acting element analysis showed that except for UGT73P12,the other five promoters of KEG-BIOs in G.uralensis contained cis-acting elements that could bind to bZIP transcription factors.It suggests that CHS,CHI,SQS,β-AS,and GuCSyGT may be the target genes binding to GubZIPs.3.GubZIPs can regulate gene expression by binding to ABRE elements in KEGBIOs.The Y1H results revealed that the GubZIP5 transcription factor could bind to the first ABRE element(GuCSyGT_ABRE1)in the promoter of the GuCSyGT gene,and the GubZIP33 transcription factor could bind to the ABRE element in the promoter of the βAS and SQS genes to regulate their expression.GubZIP1 and GubZIP56 did not bind to CHI_G-box,SQS_ABRE,β-AS_ABRE,GuCSyGT_ABRE1,and GuCSyGT_ABRE2 in this experiment,suggesting that they may not be involved in the regulation of CHI,GuCSyGT,β-AS,and SQS.4.Construction of prokaryotic expression system for GubZIPs and obtaining the soluble target protein.The prokaryotic expression system pET28a-GubZIPs and pET32a-GubZIP56 were constructed and expressed in Rosetta(DE3)cells.The cultivation condition at 16℃ and 110 rpm for 10 h was more conducive to the expression of soluble recombinant proteins pET28a-GubZIP1,and the cultivation condition at 28℃ and 110 rpm for 5 h was more conducive to the expression of soluble recombinant protein pET28a-GubZIP5.Both pET28a-GubZIP33 and pET32a-GubZIP56 could express soluble target proteins at 16℃and 20℃/28℃.The prokaryotic expression system of GubZIPs should be optimized to lay a foundation for further purification of GubZIPs protein and functional research.In this study,we analyzed the expression patterns of ABA-responsive GubZIPs and KEG-BIOs of G.uralensis,and analyzed the molecular mechanism of ABA-responsive GubZIPs regulating active components biosynthesis in G.uralensis.It is of great significance to further construct the regulation network of active components biosynthesis in G.uralensis.