| Licorice is one of the most-frequently-used Chinese herbs,which was firstly recorded on Sheng nong ben cao jing and widely used for more than 2000 years in traditional Chinese medicine(TCM)for their effects of nourishing qi,tonifying spleen and stomach,eliminating phlegm,relieving coughing,and alleviating pain.The minimum content of liquiritin(C21H22O9)in licorice slices is 0.50%stipulated in Chinese Pharmacopoeia.However,the primary investigation found that about 60%cultivated licorice didn't meet the requirement of Chinese Pharmacopoeia,which seriously affects the quality of cultivated licorice.Therefore,in this study we applied X-ray to irradiate the licorice seeds for obtaining more beneficial mutations.On the one hand,the gene polymorphisms of CHS and CHI,the two functional genes involved in the biosynthetic pathway of flavonoids in Glycyrrhiza uralensis,were analyzed.On the other hand,the transcriptome data was investigated,and five metabolic pathways influencing the biosynthesis of flavonoids were summarized.The differentially expressed genes(DEGs)on the corresponding pathways were selected and verified by qRT-PCR.In this study,licorice seeds were irradiated by six gradient doses of X-rays and cultivated for one year.One hundred and eighty-eight licorice samples were obtained,and the contents of four major flavonoids,liquiritin,isoliquiritin,liquiritigenin,and isoliquiritigenin,were assayed by HPLC.Five irradiated licorice samples with high contents of flavonoids and five blank samples with low contents of flavonoids were used as experimental materials for further genetic analysis.The gene polymorphisms of CHS and CHI were analyzed by RT-PCR,and the major haplotypes in the groups with high/low flavonoid content were determined.The major haplotypes were analyzed by bioinformatics tools.Two irradiated samples with high flavonoid content and one blank sample with low flavonoid content were used for transcriptome analysis.The DEGs on the five metabolic pathways influencing the biosynthesis of flavonoids were obtained and verified by qRT-PCR.The results of this paper are as follows:(1)Effects of X-ray on the accumulation of flavonoids in licorice:The contents of the four major flavonoids,liquiritin,isoliquiritin,liquiritigenin,and isoliquiritigenin,in 188 licorice samples were assayed by HPLC.The contents and yields of liquiritin and isoliquiritin in most of irradiated samples were significantly higher than that in the blank group,which showed an increasing-decreasing-increasing pattern with the increase of radiation dose.The yield of flavonoids in the group irradiated by 50 Gy was optimal.Five irradiated samples with high contents and yields of flavonoids,A6-1,B6-2,B6-4,C1-1,and C3-4,and five blank samples with low contents and yields of flavonoids,AO-2,B0-1,B0-3,CO-2,and CO-3,were selected to analyze the gene polymorphisms.(2)The polymorphism of CHS in licorice:One hundred and fifteen CHS cDNA sequences were obtained from the irradiated samples B6-2,B6-4,and C1-1,and blank samples B0-1,B0-3,and CO-2.The length of these CHS cDNA sequences is 1175 bp with a complete open reading frame,encoding 389 amino acid residues.Ninety-one CHS haplotypes and sixty-five CHS amino acid sequence types were determined.There were 54 haplotypes in the blank group.The haplotype-24 encoding AA-13 was the major haplotype in the blank group.There were 37 haplotypes in the irradiated group.The haplotype-61 encoding AA-47 was the major haplotype in the irradiated group.In addition,haplotype-54 encoding AA-48 also appears frequently in this group The amino acid variation analysis revealed that the V/I mutation at site 193,the I/V mutation at site 229,and the I/V mutation at site 383 appeared in the amino acid sequences may influence the accumulation level of flavonoids.(3)The polymorphism of CHI in licorice:One hundred and seventy-three CHI cDNA sequences were obtained from the irradiated samples A6-1,B6-2,B6-4,C1-1,and C3-4,and blank samples AO-2,B0-1,B0-3,and CO-3.The length of these CHI cDNA sequences is 690 bp with a complete open reading frame,encoding 229 amino acid residues.One hundred and eleven CHI haplotypes and eighty-nine CHI amino acid sequence types were determined.There were 47 haplotypes in the blank group.The haplotype-68 encoding AA-57 was the major haplotype in the blank group.There were 67 haplotypes in the irradiated group.The haplotype-3 encoding AA-3 was the major haplotype in the irradiated group.The D/E mutation at site 82 of the CHI may be related to the accumulation of flavonoid.(4)The bioinformatics analysis of the specific CHS amino acid sequences:AA-13 was the major type of amino acid sequence in the blank group.AA-47 and AA-48 were the major types of amino acid sequence in the irradiated group.The bioinformatics analysis showed that the physicochemical properties of AA-13,AA-47,and AA-48 were similar.The secondary structure and tertiary structure of AA-13 were similar to AA-47 and different from AA-48.The protein CHS is located outside the membrane without signal peptides and transmembrane domain.CHS is not a secreted protein and remains in the cytoplasm to catalyze the synthesis of flavonoids.Homology analysis indicated that CHS had a clear discrimination between different species.Protein binding site analysis showed that the 383-valine in AA-47 was a protein binding site for coumaroyl CoA.The 229-valine in AA-48 was also a protein binding site for malonyl-CoA.However,the 229-isoleucine and 383-isoleucine in AA-13 were not the protein binding sites.(5)The bioinformatics analysis of the specific CHI amino acid sequences:AA-57 was the major type of amino acid sequence in the blank group.AA-3 was the major type of amino acid sequence in the irradiated group.The bioinformatics analysis showed that the physicochemical properties of AA-3 were similar to AA-57.The secondary structure and tertiary structure of AA-3 were similar to AA-57.The CHI protein is located outside the membrane without signal peptides and transmembrane domain.CHI is not a secreted protein and remains in the cytoplasm to catalyze the synthesis of flavonoids.Besides,homology analysis indicated that CHI had a clear discrimination between different species AA-3 is aspartic acid at site 82.AA-57 is glutamic acid at site 82.Protein binding site analysis predicted that the site 82 in AA-3 and AA-57 was not the binding site of substrate.(6)The transcriptome analysis results of Glycyrrhiza uralensis samples:C3-4 and B6-4 were selected from the irradiated licorice group with high flavonoid content and BO-1 was selected from the blank group with low flavonoid content,which were renamed H1,H2,and L1,respectively.The transcriptome data of the three samples were obtained with high correct rate and good genomic coverage.More than 3795 new transcripts were obtained,which greatly enriched the gene bank of licorice.The gene expression analysis showed that three samples were different from each other.H1 vs L1,H2vs L1,obtained 4527 and 4230 DEGs,respectively.And 1875 core differentially expressed genes were got in the two comparison groups.The expression pattern of the core DEGs in samples H1 and H2 were similar and in contrast with sample L1.Sample L1 expressed more up-regulated genes than samples HI and H2.Therefore,X-ray irradiation influenced the expression level of genes,resulting in the difference in flavonoids accumulation in licorice.The core DEGs in the flavonoid metabolism pathway,terpenoid backbone biosynthetic pathway,plant hormone signal transduction pathway,plant circadian rhythm pathway,and starch and sucrose metabolism pathway,are closely related to the biosynthesis of flavonoids.The core DEGs on the five metabolic pathways are as follows:Twenty-three DEGs were obtained on the flavonoid metabolic pathway.There are ten down-regulated genes in samples H1 and H2,including one phenylalanine ammonia-lyase gene(PAL),one beta-glucosidase gene,one caffeic acid 3-O-methyltransferase gene(COMT),two coniferyl-aldehyde dehydrogenase genes(REF1),and five peroxidase genes(POD).Phenylalanine ammonia-lyase is a key enzyme on the biosynthesis pathway of phenylpropanoid.The down-regulated expression of PAL in irradiated samples H1 and H2 indicated that X-ray irradiation inhibited the enzyme function and affected the flavonoid synthesis.In addition,the expression levels of the two chalcone synthase genes in sample H1 was significantly up-regulated,which greatly influence the biosynthesis of flavonoids.The expression level of beta-glucosidase gene,REF1,and POD in the downstream alternative pathway was down-regulated,indicating that most of the reaction substrates on the phenylpropanoid metabolic pathway flew to the flavonoid synthesis pathway.Therefore,the accumulation of flavonoids in the irradiated samples was greater than that in the blank sample.Nine DEGs were obtained on the terpenoid backbone biosynthetic pathway.There are five up-regulated genes in samples H1 and H2.9-cis-Epoxycarotenoid dioxygenase gene(NCED)influenced the flavonoids accumulation.One gibberellin 2-oxidase gene(GA2ox)and two gibberellin 3-?-dioxygenase genes(GA3ox)were up-regulated,which regulated plant photosynthesis and plant primary metabolism to influence the flavonoids accumulation.One 1-deoxy-D-xylulose-5-phosphate synthase gene(DXS)was up-regulated to promote the biosynthesis of monoterpene,diterpene and carotenoids.In addition,capsanthin synthase gene(CCSI)was down-regulate in samples H1 and H2,which was beneficial to the abscisic acid synthesis and promoted the flavonoids accumulation.Eighteen DEGs were obtained on the plant hormone signal transduction pathway.There were three up-regulated genes in samples H1 and H2,one protein transport inhibitor response 1 gene(TIR1),one gibberellin receptor gene(GID1)and one jasmonate methyl-domain protein 5 gene(JAZ).The expression level of TIR1 and GID1 was up-regulated,promoting the protein ubiquitination and diterpene biosynthesis.The up-regulated expression of JAZ induced stem maturation and stress resistance,indicating that X-ray irradiation accelerated plant maturation and promoted plant stress resistance.There were ten down-regulated genes in samples H1 and H2,four SAUR-like auxin-responsive protein genes(SAUR),one auxin-reactive GH3 family protein gene(GH3),three indoleacetic acid-induced protein 10 genes(IAA10),one histidine-containing phosphotransfer factor 5 gene(AHP),and one response regulator 4 gene(A-ARR).The down-regulated expression of SAUR,GH3,and IAA10 indicated a down-regulation of auxin metabolism.AHP and A-ARR were down-regulated,resulting in a down-regulation of cytokinin metabolism.Auxin and cytokinin were both down-regulated,and then the growth rate of irradiated plants was slow down,which reflected the premature state of irradiated plants.According to the growth-differentiation balance hypothesis,the optimum defense hypothesis,and the resource availability hypothesis,slow growth contributes to the down-regulation of primary metabolism and up-regulation of secondary metabolism,which promotes the biosynthesis of flavonoids.Seven up-regulated DEGs were obtained on the plant circadian rhythm pathway of irradiated samples H1 and H2,one chalcone synthase gene(CHS),three pseudo-responsive regulator 5 genes(PRR5),two protein flowering locus T genes(FT),and one MYB-related transcription factor gene(LHY).CHS and PRR5 are related to the effect of anti-X-ray irradiation and feedback regulation of licorice.FT and LHY are related to the flowering regulation of plants.The X-ray irradiation advanced the flowering period and influenced the anthocyanids accumulation,resulting in flavonoids biosynthesisFour DEGs were obtained on the starch sucrose metabolic pathway.1,4-a-glucan branching enzyme gene(GEB)and ?-amylase gene(?-AL)were up-regulated in samples H1 and H2,promoting the production of dextrin and maltose.?-amylase gene and sucrose synthase gene were down-regulated in samples H1 and H2,respectively.The former is beneficial to the conversion of starch to dextrin and maltose,and the latter inhibits the synthesis of sucrose.In summary,a large amount of polysaccharide transform into the monosaccharide in irradiated licorice,providing a basis for the formation of glycosides.(7)Analysis of relative expression levels of core DEGs in the transcriptome of Glycyrrhiza uralensis:Twelve core DGEs were verified by Real-time PCR.They were PAL(Glyur000106s00011717),COMT(Glyur000116s00009246),CHS1(Glyur000424s00026890),CHS2(Glyur006062s00044203),NCED(Glyur000278s00017280),GA2ox(Glyur000261s00014360),DXS(Glyur000231s00022061),GID1(Glyur000158s00011331),JAZ(Glyur002299s00036262),SA UR(Glyur000017s00002448),LHY(Glyur000116s00009244),and AMYB(Glyur000047s00004005).Except DXS and COMT,the expression level of other genes was consistent with the RNA-Seq results. |
PDF Full Text Request