The Literature Analysis Of Aconitum Poisoning And Protein Expression Of Candidate BAHD Determing Acute Toxicity Of A.carmichaelii

Aconitum plants are important traditional chinese herbal medicines,it have significant cardiac,analgesic,anti-inflammatory and other pharmacological effects,which had been applicatied in long chinese history,however,due to the acute toxoity that make the application of Aconitum plants were severely limited.Aconitum toxicity can be reduced by concocting and other methods,but cannot reduce the toxicity completely.For a long time,with the tradition of food therapy for disease by using Aconitum in the folk,aconitum poisoning incidents occurred frequently in various parts of our country.Through the analysis of literature,this study counted a total of 5593 cases of aconitum poisoning,which mainly occurred in Yunnan?38.62%?and Sichuan?10.37%?provinces.The main causes of poisoning were drinking medicinal liquor?20.17%?and treatment and food therapy?13.82%?.Exploring reduces toxicity meanwhile preserves efficacy and ensuring the safety,effectiveness,and control of Aconitum is a problem that needs to be solved urgently.Diterpenoid alkaloids are important basic substance for pharmacology of Aconitum plants,the C₈ acetyl ester and C₁₄4 benzoyl ester on the skeleton are closely related to the acute toxicity of Aconitum,and BAHD Acyltransferases,which catalyzes the synthesis of ester groups,are the key end enzymes in alkaloid skeleton of aconitum caryophyllus.Therefore,finding and functional identification the major BAHD genes are important process to solve the toxicity problem of Aconitum.By prokaryotic expression and in vitro enzyme assay,the candidate BAHD genes were functionally identification from Aconitum carmichaelii,which the representative species of Aconitum.The major results were as follows:In this study,six key candidate genes of Aconitum BAHD acyltransferase were screened,constructured candidate genes to the prokaryotic expression vector pGEX4T-1 and transformed these plasmid expression host strain BL21?DE3?,obtained different recombinant expression vectors and heterologous expression strains,respectively.The recombinant proteins were successfully induced by using the E.coli expression system.The optimal conditions were28°C,4 h,IPTG 0.5 mM.Acyl donors acetyl-CoA and benzoyl-CoA,acyl acceptor benzoylaconine,benzoylmseaconine,benzoylhypaconine and aconine as substrates,using heterologously induced proteins for enzymatic reactions,but none of the products were detected.This study induced the Heterologous protein of the key candidate BAHD gene from Aconitum carmichaelii,it laid the foundation for the identification of other candidate genes and the key BAHD genes determine the acute toxicity of Aconitum carmichaelii,in order to solve the toxicity of Aconitum and to create new germplasm resources with low toxicity and high efficiency.