Identification And Function Of Key BAHD Acyltransferase Genes Determing The Acute Toxicity Of Aconitum L.

Aconitum L.,one of the main highly toxic medicinal plants of the Ranunculacea,has excellent analgesic and anti-inflammatory functions.The main active and toxic substances of Aconitum are diterpenoid alkaloids(DAs),and the toxic characteristics of Aconitum are closely related to the C-8 and C-14 ester groups on the DAs skeleton.The BAHD acyltransferase is a key terminal enzyme for the synthesis of ester groups,indicating that BAHD acyltransferase is closely related to the formation of the toxicity of Aconitum.Therefore,the functional study of the key BAHD acyltransferase gene is particularly important in solving the toxicity of Aconitum.In this study,toxic Aconitum carmichaelii and Aconitum vilmorimianum with rich esters,and non-toxic Aconitum weixiense without esters were used as the main component.The BAHD acyltransferase genes from three species of Aconitum were cloned and analyzed by bioinformatics.According to the BAHD family members in the phylogenetic tree,characterization of the spatio-temporal expression of BAHD candidate genes in different developmental stages of Aconitum carmichaelii,the candidate BAHD genes of Aconitum carmichaelii were screened responsible for the formation of toxicity.The main results are as follows:1.The 24,22 and 17 BAHD gene family members were cloned from Aconitum carmichaelii,Aconitum vilmorimianum and Aconitum weixiense respectively.Most of the BAHD sequences contained two conserved domains of HXXXD and DFGWG.A few did not contain HXXXD motif.BAHD proteisn are hydrophilic protein,there is no transmembrane structure,and the entire N-terminus does not contain a signal peptide,such proteins located in the cytoplasm.Therefore,the biosynthesis of ester groups presumably located in the cytoplasm.2.In the BAHD phylogenetic tree,the members of the branch of the ? subgroup mainly used acyl-CoA as the acyl donors and they involved in the acylation of the alkaloid.While the members of the Va subfamily used hydroxycinnamoyl/benzoyl-CoA as the acyl donor,indicating that the members of the two branches are closely related to the toxicity of Aconitum.In this study,11 ? branches and 5 Va members were obtained from Aconitum carmichaelii,9? branches and 3 Va members from Aconitum weixiense.There were 11 ? branches and 3 Va members of Aconitum vilmorimianum.The The results showed that there were differences between the base sequence and the amino acid of the BAHD genes obtained from the same pair of primers from three Aconitum species.The BAHD genes from Aconitum weixiense can not encode functional acyltransferases or a low activity of the encoded acyltransferases,resulting in low toxicity of diterpene alkaloids.3.The relative expression of BAHD acyltransferase gene were detected by relative quantification(qRT-PCR).The results showed that the expression of BAHD gene in stem and root was low and did not change with the development degree.The expression patterns were similar in the leaves and lateral roots,and gradually increased from April to June,and then decreased gradually from July to September.DAs are mainly concentrated in the lateral roots.The expression of BAHD genes in the lateral roots of AcBAHD4,AcBAHD5,AcBAHD6,AcBAHD7,AcBAHD14,AcBAHD17 and AcBAHD26 is high,which indicates that the above seven BAHD genes may be involved in diterpene alkaloid Base biosynthesis.AcBAHD4,AcBAHD6,AcBAHD8,AcBAHD9,AcBAHD12,ACBAHD13,AcBAHD14,AcBAHDl6,AcBAHD17,AcBAHD19,AcBAHD20,AcBAHD21,AcBAHD24 were selected as candidate genes closely related to the toxicity of Aconitum.The BAHD gene of the non-toxic Aconitum weixiense was also expressed in all tested organs,but the diterpenoid alkaloids in Aconitum weixiense did not produce esterification,suggesting that the low activity of BAHD acyltransferases in Aconilum weixiense or lost of function after modified.