Bioinformatics Analysis And Prokaryotic Expression Of CAD Gene In Asarum Sieboldii Miq.

Cinnamyl alcohol dehydrogenase(CAD),which is one of the key enzymes in biosynthesis pathway of phenylpropanoid,can reduce the cinnamaldehyde to the corresponding cinnamyl alcohol.Volatile oil is the main efficacy material of Asarum,and methyl eugenol and other aromatic compounds,which are the main components of volatile oil,are all based on phenylpropanoid metabolic pathway.Therefore,the separation and functional verification of Asarum cad gene is theoretical basis to clarify the molecular regulation mechanism of the biosynthesis pathway of main active ingredients in Asarum,to conduct genetic improvement of Asarum,to enhance quality of Asarum.Based on the principle and method of homologous cloning,the cad gene sequence was obtained.In this study,the on-line analysis tools and application software were used to analyze the bioinformatics of cad gene encoding protein,to understand the basic physicochemical properties and functional structure information.of the encoded protein.At the same time,the recombinant plasmid was constructed by TOPO cloning and LR reaction.The recombinant plasmid was successfully transfected into E.coli BL21 by colony PCR and sequencing.The expressed protein was induced by IPTG.The protein was purified by affinity chromatography and was analyzed by solubility,SDS-PAGE,Western Blot and ELISA.The results of bioinformatics analysis showed that the open reading frame of the cad gene encodes a total of 356 amino acids.The basic physical and chemical properties showed that the coding protein was acidic,hydrophilic and steady.The structural analysis showed that the protein family was an alcohol dehydrogenase superfamily with a zinc finger structure.The domain includes a GroES-like region,a NAD(P)binding domain,a polyketide synthase domain and an enoylreductase domain,and there was a more obvious coiled coil structure at 300-320 amino acids.The secondary structure type is α-β and spiral and fold are the main structural elements.The subcellular localization and signal peptide analysis showed that the protein was non-secreted protein.The Sequence homology comparison results showed that the homology of this protein was 76% with that of grape.The prokaryotic expression results showed that the recombinant plasmid pDEST17-cad was successfully constructed and and the protein was soluble and non-secreted.The analysis of SDS-PAGE and Western Blot showed that the recombinant plasmid pDEST17-cad was successfully expressed in Escherichia coli and and the purified His-CAD fusion protein was successfully obtained.The results of protein expression level at different induction time points showed that the expression level of protein was gradually increased with time,however,after 16 hours,the amount of protein expression was decreased and the protein expression level tends to saturation.In the present study,the bioinformatic analysis and prokaryotic expression analysis of the obtained cad gene were used to study the regulation mechanism of Aspergillus flavonoids biosynthesis,as well as the genetic improvement and secondary metabolism.The implementation of the project provides a theoretical basis,with a positive theoretical and practical significance.