Exploration And Functional Identification Of Key Enzyme Genes Of Aristolochic Acid Synthetic Pathway In Asarum

Asarum is able to disperse chill cold imposed on the body and cold produced in vivo,and has analgesic and antitussive effects at the same time.It is an important medicine for treating wind chill,headache and cough.Asarum belongs to aristolochiaceae which contains aristolochic acid.At present,many kinds of Chinese herbal medicines containing aristolochic acid are banned or restricted in use.Traditional Chinese medicine methods like processing,cooking and reasonable compatibility have a certain effect on the removal of aristolochic acid,but it is difficult to fundamentally solve the problem of aristolochic acid toxicity.The continuous advancement of biotechnological methods has opened up a new world for the attenuation of traditional Chinese medicines.It is necessary and possible to understand how to master the synthetic pathway of aristolochic acid in plants by modern biotechnology,and to remove or reduce aristolochic acid in Asarum at the genetic level from the perspective of genetics.The study was conducted in response to the above problems.In this study,no-parameter high-throughput sequencing was first used to obtain information on the full-length transcriptome of the Northern Asarum.The transcriptome sequencing results were synthetically analyzed,and the metabolite of cepharanthine which is closest to aristolochic acid in the biosynthesis pathway of aristolochic acid was found in KEGG pathway map named 350.After synthesizing the up-regulated and down-regulated genes in KEGG pathway map named 950,the tyrosine decarboxylase family gene was identified as a key enzyme gene in the aristolochic acid biosynthesis pathway,and further research was conducted in this study.In this study,TyrDC1,TyrDC2 and TyrDC3 were successfully cloned.The sequencing results were compared on BLAST and identified as complete CDS gene sequences.During base sequence comparisons of TyrDC1 and TyrDC3,genes of other species were found to have homology higher than 90% with TyrDC1 and TyrDC3.But TyrDC2 did not have high homologous genes.It had about 60% homology similarity with aromatic-L-amino acid decarboxylase predicted protein.By phylogenetic tree and base sequence comparisons,TyrDC2 was determined to be a TyrDC that has not been reported in plants including Asarum.The subcellular localization of TyrDC1,TyrDC2 and TyrDC3 were predicted and the proteins were identified as functional proteins.The secondary and tertiary structures of TyrDCs were predicted.TyrDC1 and TyrDC3 were non-stable proteins and TyrDC2 was a stable protein.Plant Expression Vector of PRI101-TyrDC1,-TyrDC2 and-TyrDC3 was successfully constructed.And the genetic transformation system of callus of northern Asarum was established.Recombinant plasmid was transformed into the genetic transformation system of Asarum.The system was evaluated to be relatively stable and have high conversion efficiency,which can lay the foundation for downstream molecular experiments.HPLC analysis between the transgenic northern Asarum and the control group was performed.The results showed that the content of aristolochic acid A in the transgenic callus was higher than that in the control group(P<0.05),indicating that TyrDCs can promote the biosynthesis of aristolochic acid A(AAI).Prokaryotic expression vector of pEASY-Blunt E1-TyrDC1,-TyrDC2 and-TyrDC3 were successfully constructed and could express soluble protein.Western blot experiments confirmed that the expressed protein is TyrDC.The validation of the expressed protein in vitro confirmed that TyrDCs had a significant effect on the content of AAI in the grinding liquid of Asarum leaves.The efficiency of the protein extract mixture was the highest,and the effect of TyrDC2 was higher than that of the other two species.There was a significant difference compared with the AAI in the control group(P<0.05).Silencing Expression Vector of pTRV-2-TyrDC1,-TyrDC2 and-TyrDC3 was constructed.Injection infection of the leaves of Asarum was conducted,and the leaves’ color turned lighter and shrinkage,and the vein turned protruding.The qRT-PCR data showed that the relative expression levels of TyrDC1,TyrDC2,and TyrDC3 were decreased in the infected plants.HPLC data of the infected plants also showed that the content of AAI was relatively decreased.This two data are consistentAmong the TyrDCs,TyrDC2 has the greatest influence on the biosynthesis of AAI.We inferred that TyrDC2 may be a unique enzyme in the plant containing aristolochic acid,which may play a more critical role in the biosynthesis of aristolochic acid.