Cloning And Expression Of HMGR And CHS Genes In Polygala Tenuifolia Willd

ObjectiveCloning 3-hydroxy-3-methyl glutaryl coenzyme A reductase（HMGR）and chalcone synthase（CHS）gene in Polygala tenuifolia.Analyze the effect of gene expression on the content of medicinal ingredients,and verify the function of HMGR from the perspective of enzyme activity.To provide reference for the sustainable use of resources and germplasm improvement of P.tenuifolia.Methods（1）The full-length sequence of the HMGR and CHS genes of P.tenuifolia were cloned by rt-pcr combined with RACE,and perform bioinformatics analysis on the sequence.At the same time,the function and structure of the protein encoded by HMGR and CHS genes were predicted and analyzed.（2）The expressions of HMGR gene and CHS gene,total saponins and total flavonoids in different parts of roots,stems and leaves of P.tenuifolia were determined by RT-qPCR,UV spectrophotometry and high performance liquid chromatography,respectively.The correlation between gene expression and medicinal components was analyzed.（3）The function of PtHMGR was verified by gas chromatography-mass spectrometry.Results（1）The full-length sequences of HMGR and CHS genes in P.tenuifolia were cloned and named PtHMGR and PtCHS,respectively（The login Numbers of GenBank are MK424118 and MK424117 respectively）.The cDNA sequence of PtHMGR was 2323 bp,including a complete open reading frame of 1782bp,encoding 593 amino acids.The physical and chemical properties of the protein encoded by PtHMGR gene were predicted:the molecular formula was C₂₇₈₁H₄₄₈₆N₇₇₀O₈₅₃S₃₃,the molecular weight was 63414.84 Da,and the isoelectric point PI was 6.09.The cDNA sequence of PtCHS was 1634 bp,including a complete open reading frame of 537 bp,encoding 178 amino acids.The physical and chemical properties of the protein encoded by PtCHS gene were predicted:the molecular formula was C₈₉₅H₁₄₂₀N₂₄₄O₂₅₁S₁₂,the molecular weight was 19999.33 Da,and the isoelectric point PI was 9.25.（2）PtHMGR and PtCHS genes were differentially expressed in roots,stems and leaves.Among them,PtHMGR gene had the highest expression in roots,followed by leaves and lower expression in stems.The relative expression of PtCHS gene was highest in stems,followed by leaves and roots.There are also differences in the contents of total saponins,total flavonoids,tenuifolin and polygalaxanthone III in roots,stems and leaves of P.tenuifolia.The total saponins,tenuifolin and polygalaxanthone III were higher in the roots,while the total flavonoids mainly accumulated in the stems.Analysis of the correlation between the expression levels of PtHMGR and PtCHS genes and medicinal components showed that the content of total saponins and tenuifolin was positively correlated with the expression of PtHMGR gene;The content of total flavonoids was positively correlated with the expression of PtCHS gene,while the content of polygalaxanthone III was negatively correlated with the expression of PtCHS gene.（3）PtHMGR was successfully expressed after the induction of IPTG,and catalyzed the3-hydroxy-3-methyl glutaryl coenzyme A（HMG-CoA）to generate mevalonolactone.Conclusion（1）In this study,the full sequence of the HMGR and CHS genes was cloned from P.tenuifolia for the first time.Analysis of the correlation between the expression levels of PtHMGR and PtCHS genes and medicinal components showed that the content of total saponins and tenuifolin was positively correlated with the expression of PtHMGR gene;The content of total flavonoids was positively correlated with the expression of PtCHS gene.The function of PtHMGR was verified by gas chromatography-mass spectrometry.The results showed that PtHMGR has the catalytic function of HMGR enzyme,and catalyzed the 3-hydroxy-3-methyl glutaryl coenzyme A（HMG-CoA）to generate mevalonolactone.Therefore,it is possible to promote the accumulation of the pharmacological components of P.tenuifolia by up-regulating the expression of the gene,thereby improving the quality of the P.tenuifolia.