| Licorice is one of the most frequently-used Chinese herbs with the functions of heat-clearing and detoxicating,spleen and supplement Qi,alleviating pain,relieving coughs,and moderating the property of herbs.In addition,licorice is also widely used in the field of food and cosmetics industry.Due to the excessive collection of wild licorice,digging wild licorice has been explicitly prohibited by China's state council in 2000.At present,the cultivated licorice become the main source of commercial licorice.However,in our previous research,the quality of cultivated licorice is generally lower than wild licorice.How to improve the quality of cultivated licorice has been a key issue restricting the sustainable development of licorice resources.Glycyrrhizic acid is one of the marker components in Chinese Pharmacopoeia 2015.Our previous study found that Glycyrrhiza glabra contained the highest level of glycyrrhizic acid in the three original plants of licorice,which demonstrates that G.glabra is an excellent experimental material to study the molecular regulatory mechanism of glycyrrhizic acid biosynthesis.X-ray irradiation treatment is simple and convenient with a high mutation frequency and a wide mutation range,which has been used in plant breeding.Therefore,based on the X-ray irradiation treatment of G.glabra,samples with the most significant difference of the contents/yields of glycyrrhizic acid were selected.RNA-Seq sequencing technology was used to construct differential gene expression maps for screening key regulatory genes for glycyrrhizic acid biosynthesis.This paper will lay a foundation for molecular breeding and screening better germplasm resources of licorice.In this paper,X-ray irradiation was used to treat the seeds of three original plants of licorice,and the contents of 18a-glycyrrhizic acid and 18?-glycyrrhizic acid were assayed by HPLC,and the yields were calculated.Three high glycyrrhizic acid contents/yields irradiated G.glabra samples and one low glycyrrhizic acid content/yield blank G.glabra sample were selected for RNA-Seq sequencing.The new transcript was predicted,alternative splicing and variant of point were obtained,gene function was annotated by means of bioinformatics analysis.Differential expression analysis was used to obtain 1736 mutual differentially expressed genes(DEGs),and 18 candidates key functional genes were screened from the result of gene function enrichment.The transcriptome data were verified by qRT-PCR.An auxin-responsive protein IAA gene and an UDP-glucose 4-epimerase gene were successfully cloned,then the plant binary expression vectors containing these two genes were successfully constructed,which laid a foundation for further establishment of gene overexpression system.The results of this paper are as follows:(1)Effects of X-ray on the contents and yileds of triterpenoids in G.glabra:The contents of 18a-glycyrrhizic acid and 18,8-glycyrrhizic acid in 159 licorice samples were determined by HPLC and the yields were calculated.The results showed that the contents and yields of 18a-glycyrrhizic acid and 18?-glycyrrhizic acid in most of the irradiated licorice samples were significantly higher than that in the blank control samples.Therefore,X-ray irradiation treatment increased the biomass of the licorice samples and improved the accumulation of 18?-glycyrrhizic acid and 18?-glycyrrhizic acid.Three high glycyrrhizic acid contents/yields G.glabra samples HI,H2 and H3 and one low glycyrrhizic acid contents/yields blank control sample L1 were selected for further RNA-Seq sequencing.(2)The transcriptome analysis of G.glabra samples with differential triterpenoids accumulation:The transcriptome data of four samples were firstly obtained with a mass of alternative splicing and good genomic coverage.10149 new transcripts were obtained and a large number of structural genes were optimized,which greatly expanded the gene library of licorice.As a result of GO and KEGG annotation,the GO database annotated 13598 functional genes,KEGG annotated 9647 functional genes,and successfully co-annotated 16733 functional genes.56 genes were identified as the key genes directly involved in the biosynthetic pathway of glycyrrhizic acid.Through gene differential expression analysis,the three comparison groups,L1-H1,L1-H2,and L1-H3,obtained 3955,3906,and 3478 differential genes,respectively.And a total of 1376 co-expressed DEGs were determined.The cluster heat map of the 1376 co-expressed DEGs showed that the samples with high contents/yields of glycyrrhizic acid had a good consistency in expression pattern compared with L1.Enrichment analysis indicated that a large number of DEGs were enriched in the following five metabolic pathways,ubiquinone and other steroidal biosynthetic pathways,sugar metabolism pathway,phytohormone signal transduction pathway,phenylpropanoid metabolism pathway,and plant circadian rhythm pathway.Eighteen candidate key functional genes were screened from these pathways,including HMGR,CRTZ,GA2ox,AMYB,glgc,PP2C,ARPI,PRR5,DXS,LIS,UGE,VR,I2'H,F3'H,COMT,CHI,F5'H and CHS.(3)qRT-PCR verification of candidate key functional genes:A total of eighteen candidate key functional genes were verified by qRT-PCR,and compared with L1,the expression of eight DEGs was up-regulated and the other ten DEGs was down-regulated.The result of correlation analysis between qRT-PCR and RNA-Seq indicated that they had relative similarity with each other.The Auxin-responsive gene ARPI and UDP-glucose 4-epimerase gene UGE were selected from 18 candidate functional genes for gene cloning and expression vector construction.(4)Cloning and bioinformatics analysis of GgARPI gene from G.glabra:According to the genomic information of licorice,a 686 bp cDNA sequence of GgARPI gene was successfully amplified by PCR with the specific primers.The open reading frame is 585 bp,encoding 194 amino acid residues.The results of bioinformatics analysis indicated that ARPI was a stable hydrophilic protein.We successfully predicted its secondary and tertiary structure.The protein does not contain signal peptide or transmembrane domain.In addition,homology analysis indicated that GgARPI had a clear discrimination between different species.It showed a closer evolutionary relationship with leguminous plants.(5)Cloning and bioinformatics analysis of GgUGE gene from G.glabra:According to the genomic information of licorice,the 1121 bp cDNA sequence of GgUGE gene was successfully amplified by PCR with the specific primers,The open reading frame is 1053 bp,encoding 350 amino acid residues.The results of bioinformatics analysis indicated that UGE was a unstable hydrophilic protein.We successfully predicted its secondary and tertiary structure.The protein does not contain signal peptide or transmembrane domain.In addition,homology analysis indicated that GgUGE had a clear discrimination between different species.It showed a closer evolutionary relationship with leguminous plants.(6)Construction of plant binary expression vectors containing GgARPI or GgUGE:The target gene fragment was ligated with the digested vector pCAMBIA1305.1 by effusion kit to construct the binary over-expression vectors containing GgARPI or GgUGE.The successfully constructed vectors pCA-GgARPI and pCA-GgUGE were transformed into E.coli.PCR and sequencing were used to detect the validity of the insertion. |
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