Isolation,Expression Analysis And Function Characterization Of CAD Gene Of Asarum Sieboldii Miq

Methyleugenol is important volatile oil and pharmacodynamic component of traditional Chinese medicine Asari Radix et Rhizoma.Cinnamyl alcohol dehydrogenase(CAD)catalyzes coniferyl aldehyde to produce coniferyl alcohol in biosynthetic pathways of methyleugenol.So,to study the biosynthetic mechanism of methyleugenol,it is important to isolate CAD genes and verify its function.In this study,seven putative CAD genes(AsAD1-7)were selected from the transcriptome library,and one(AsCAD)was selected through homologous cloning.We predicted the characteristics of eight CAD family members of Asarum sieboldii by bioinformatic techniques.The most suitable reference gene 18SrRNA was selected by real-time fluorescence quantitative PCR.Then we analyzed expression profile of AsCADs in different tissues and phases in the same way.To study the function of CAD gene,we used AsCAD as the object gene to construct the overexpression vector pK7FWG2.0-AsCAD by Gateway technique,transform it in the model plant Arabidopsis thaliana,and measure the lignin content to verify the function of AsCAD gene.The main findings are as follows:(1)Based on the transcriptome library,seven members of Asarum sieboldii CAD gene family(AsCAD1-7)was screened,having GenBank access numbers from KY446440-KY446446 respectively.The AsCADs were analyzed by bioinformatics software and the results showed that the molecular weight of AsCAD 1-7 was from 30 kD-40 kD.Also they showed that proteins encoded by AsCAD1-7 belonged to acid labile hydrophilic proteins that did not have transmembrane structure or signal peptide.They all showed characteristics of MDR superfamily members with Zinl,Zin2 and NADPH conservative structural domains.(2)Based on the principles and method of homologous cloning,the full-length cDNA sequence of AsCAD having GenBank access number KT454711,was cloned by PCR and RACE amplification technique.Bioinformatics analysis showed that AsCAD belongs to the CAD gene family.(3)In this study,we screened the reference genes from SAND-1,ACT,18S rRNA,CYP-2,GAPDH and TUB by real-time fluorescence quantitative PCR and analysis softwares like geNorm,NormFinder,Best Keeper,Delta CT.The results showed that the expression levels of 18S rRNA was stable in different tissues(roots,rhizomes,leaves,petioles and flowers)and in different growth stages(young phase,flowering phase and maturation stage).And it can be the reference gene of differentially expression analysis of A.sieboldii genes.(4)The expression profile of AsCADs was analyzed by real-time quantitative PCR.The results showed that AsCADs were expressed in all tissues and all growth phases,but the expression level was different.AsCAD3 and AsCAD6 had the lowest expression level of all.Overall,in the same tissue,the expression level showed a trend that in young phase expression was the lowest,in flowering phase it was lower and in the maturation stage it was highest.In the same growth period,AsCADl and AsCAD had the same expression profile that the expression level of underground part(roots and rhizomes)is higher than ground part(leaves and petioles),which might be related to their function.AsCAD5 had higher expression level in roots while lower in leaves.AsCAD7's expression level have no significant difference among three periods.(5)In this study,we obtained the AsCAD overexpress transgenic Arabidopsis thaliana plants by the construction of over-expression vector pK7FWG2.0-AsCAD and the successful transformation of wild type A.thaliana Lignin content was measured,and the results showed that lignin content of transgenic A.thaliana was 3.20%higher than the wild-type,which had an increase of 32.74%.They indicated that AsCAD had enzyme activity and was involved in lignin biosynthesis.It was the first time for the CAD genes to be isolated from Asarum sieboldii and to select the most stable reference gene for differential expression analysis of Asarum sieboldii.The function of AsCAD was also verified.So the present research will underlie a significant basis not only for the further function verification of the AsCAD genes but also to the mechanism elucidation of methyleugenol biosynthesis regulation.