Cloning And Functional Verification Of Aster Tataricus Squalene Synthase Gene(AtaSQS)

Aster tataricus(L.)is a perennial herb in the Compositae family.Its dried root and rhizome are widely used as a traditional medicine to relieve cough and phlegm,antioxidation,anti-inflammation,anti-tumor,and laxative diuresis,In China,A.tartaricus is mainly distributed in Anguo City,Hebei Province,Bozhou City,Anhui Province,and the three provinces in Northeast China.Aster from Anguo,which is considered to be the best aster and was included in the National Geographic Indication Products List in 2013,has thick,long and red color roots.Aster contains abundant primary and secondary metabolites,among which the main active ingredient for relieving cough and phlegm is shionone,a triterpenoid compound.According to the Pharmacopoeia of the People’s Republic of China,shionone is a required index for evaluating aster quality,and its content should not be less than 0.15%.Understanding the metabolic mechanism of triterpenoids in aster is of great significance for improving the content of shionone.However,there are few studies on the regulation of biosynthesis of aster triterpenoids.In this study,Qiziwan was used as the material to study the spatiotemporal accumulation patterns of total triterpene and shionone,and the effects of methyl jasmonate on the total triterpene content,shionone content and genes expression levels related to triterpenoids biosynthesis in A.tataricus.The squalene synthase gene(AtaSQS),which is a key gene in the biosynthesis of triterpenoids,and its promoter were cloned.The expression pattern of AtaSQS and the cis-acting elements on its promoter were analyzed.A taSQS overexpression Arabidopsis thaliana strains were obtained.The effect of A taSQS overexpression on total triterpene content in Arabidopsis thaliana was clarified.Transcription factors that may regulate AtaSQS expression were screened out by WGCNA.The main results are as follows:1.During the development of aster,the total triterpene content showed a trend of increase-decrease-increase-decrease,and the content of shionone showed a trend of increasing first and then decreasing.There were differences in total triterpene and shionone contents in different tissues.With the highest total triterpene content in leaf and the lowest content in flower,and the highest shionone content in the root and lowest in the stem.2.Methyl jasmonate promoted the accumulation of total triterpene and shionone in aster.The total triterpene content increased significantly after 0.5 h of treatment,then gradually decreased,and returned to normal level at 9 h.The content of shionone increased significantly at 3 h of treatment,gradually decreased,and returned to normal at 9 h.The expression of 16 triterpenoids biosynthesis-related genes,including AtaHMGS1,AtaHMGS2,AtaHMGS3,AtaHMGR1,A taHMGR2,AtaMK,AtaPMK,AtaMVD1,AtaMVD2,AtaFPS1,AtaFPS2,AtaFPS3,AtaSQS,AtaSQE1,AtaSQE2,and AtaSHS,were upregulated to varying degrees under methyl jasmonate treatment.3.The CDS sequence of AtaSQS is 1257 bp,and the protein encoded by AtaSQS is an unstable hydrophilic protein containing 418 amino acids with a molecular weight of 47.88 kD.The result of transmembrane structure prediction analysis showed that the protein encoded by AtaSQS had a transmembrane helix structure at amino acids at positions 387409.The prediction result of protein secondary structure showed that the secondary structure of the protein encoded by AtaSQS was mainly α-helix and irregular curling.The result of multiple comparison of amino acid sequences showed that the squalene synthase proteins of aster and other plants had two functional regions of squalene and octahydrolycopene and two aspartic acid residue regions,which were conserved structural regions of squalene synthase.The result of phylogenetic analysis showed that the amino acid sequence of squalene synthase was more conserved in Asteraceae.Subcellular localization result indicate that the protein encoded by AtaSQS is localized to the endoplasmic reticulum.The expression of AtaSQS gradually increased with the development of aster.The expression level of AtaSQS is lower in the root.4.Arabidopsis thaliana strains of overexpressed AtaSQS were obtained and overexpression of AtaSQS upregulated the expression levels of upstream and downstream genes of Arabidopsis thaliana squalene synthase gene and increased the total triterpene content.It is explained that AtaSQS plays a key role in triterpenoids biosynthesis5.The promoter of AtaSQS was cloned.The promoter of AtaSQS contained ethyleneresponsive element,salicylic acid response element,light-responsive element,and mechanical damage response element,as well as binding sites of transcription factors such as WRKY,MYB,MYC,and ARF.It is indicated that the expression of AtaSQS may be regulated by conditions such as plant hormones,mechanical damage and light,as well as transcription factors such as WRKY,MYB,MYC,and ARF.One WRKY,6 MYB,and 2 MYC transcription factors,which are co-expressed with AtaSQS,were screened by WGCNA.It was preliminarily judged that these transcription factors may regulate the expression of AtaSQS.In summary,the spatiotemporal accumulation patterns of total triterpene and shionone of aster were revealed in this study,which provided a theoretical basis for the optimal harvest period and organs of aster.The key role of AtaSQS in the biosynthesis of aster triterpenoids was clarified,and a theoretical basis was provided for improving the quality of aster by molecular means.