Regulation Of Licorice DXS And F5H Genes On Glycyrrhizic Acid Biosynthesis

Licorice is one of the most commonly-used Chinese herbs and known as the "Guo Lao"and "Shi Fang Jiu Caol".The Chinese Pharmacopoeia stipulates that glycyrrhizic acid(GA)is one of the marker components in licorice.In our previous transcriptome analysis of Glycyrrhiza glabra,it was found that 1-deoxy-D-xylulose-5-phosphate synthase(DXS)and ferulate 5hydroxylase(F5H)genes significantly negatively influenced the biosynthesis of GA.In this paper,we further explore the negative control of these two genes on the accumulation of GA.DXS and F5H cDNA sequences were cloned from the cDNA library of G.glabra.Plant binary expression vectors overexpressing DXS and F5H genes were constructed using gene fusion methods.sgRNA sequences of the two genes were designed by online tools(www.benchling.com)and used to construct CRISPR/Cas9 vectors.Recombinant Agrobacterium rhizogenes(ATCC15834)strains containing the above vectors were constructed by electroporation.Licorice hairy root lines overexpressing and silencing DXS and F5H genes were induced by infecting licorice sterile seedlings with recombinant A.rhizogenes strains.The content of GA in each hairy root line was assayed using UPLC.The functions of DXS and F5H genes on GA biosynthesis were clarified.The following results were obtained in this paper:(1)Cloning DXS and F5H cDNA sequences from G.glabra:The DXS and F5H cDNA sequences were cloned from the cDNA library of G.glabra and transformed into E.coli DH5a competent cells.Then these sequences were uploaded to the GenBank database(Accession Number:DXS:MN158121,F5H:MK882511).(2)Construction of plant binary expression vectors:The plant binary expression vectors overexpressing DXS and F5H genes were constructed by gene fusion method with Bgl ? and Spe ? at pCAMBIA1305.1 as the insertion sites.Then the binary expression vectors were transformed into E.coli TOP 10 competent cells.(3)Construction of CRISPR/Cas9 vectors:The sgRNA sequences of DXS and F5H genes were designed using the online tool,Benchling,respectively.The CRISPR/Cas9 vectors were constructed using the plasmid pHSE401 with Bsa I as the insertion site.Then the recombinant vectors were transformed into E.coli TOP10 competent cells.(4)Construction of licorice hairy root lines overexpressing DXS and F5H genes:The plant binary expression vectors overexpressing DXS and F5H genes were transformed into ATCC15834 by electroporation and verified by PCR and sequencing.The recombinant ATCC15834 strains were used to infect licorice sterile seedlings to induce hairy root lines.The hairy roots overexpressing DXS and F5H genes were identified and screened by PCR and sequencing.(5)Construction of licorice hairy root lines silencing DXS and F5H genes:The CRISPR/Cas9 vectors were transformed into ATCC 15834 by electroporation and verified by PCR and sequencing.The recombinant ATCC15834 strains were used to infect licorice sterile seedlings to induce licorice hairy root lines.The edited hairy root lines silencing DXS and F5H genes were identified by PCR and sequencing.(6)Determination and analysis of GA content in licorice hairy root samples:The GA content in 27 licorice hairy root samples,including wild type group(1 sample),negative control group(2 samples),gene overexpressing group(17 samples)and gene silencing group(7 samples)was assayed by UPLC.Statistical analysis showed that the GA content in the silencing group was significantly higher,whereas in the overexpressing group was significantly lower than that in the wild type group and negative control group.(7)The regulation of DXS and F5H genes on the biosynthesis of GA:In this paper the functions of DXS and F5H genes were identified by gene overexpression and silencing based on reverse genetics.It was confirmed that the expression levels of DXS and F5H genes were negatively correlated with GA content,indicating their negative regulation on GA biosynthesis,which was in accordance with our previous transcriptome sequencing results.