Differential Gene Analysis Of The Asarum Heterotropoides Fr. Schmidt Var. Mandshuricum(Maxim.) Kitag.transcriptome Data And Exploration Of Key Genes In Aristolochic Acid Synthesis

Asarum heterotropoides Fr.Schmidt var.mandshuricum(Maxim.)Kitag is a common kind of Chinese medicine,and there are original plants of Asarum recorded in the 2015 edition《Chinese Pharmacopoeia》: Asarum heterotropoides Fr.Schmidt var.mandshuricum(Maxim.)Kitag,Asarum sieboldii Miq.var.seoulense Nakai,and Asarum sieboldii Miq.Asarum heterotropoides possesses antipyretic effects,could alleviate cough,diminish water swelling,calm analgesia,and relieve asthma.Asarum heterotropoides Fr.Schmidt var.mandshuricum(Maxim.)Kitag contains aristolochic acid,a phenanthrene derivative,which is an isoquinoline alkaloid,and could cause permanent kidney damage in a short time.Researches have shown that the nitro group is the main toxic group in aristolochic acid derivatives,while the nitro group in aristolochic acid derived from the amino group of aspartic acid.Aristolochic acid is found in most kinds of the genus aristolochia and asarum.Using drugs containing a high content of aristolochic acid will cause toxic and side effects.At present,the methods for attenuating the toxicity of Chinese medicine containing aristolochic acid are mainly through processing and compatibility of medicine.Studies on the molecular mechanism of detoxification of medicinal plants containing aristolochic acid has not been reported.In summary,based on the previous experiments,the differential gene was analyzed through the transcriptome data of Asarum heterotropoides Fr.Schmidt var.mandshuricum(Maxim.)Kitag,and the important candidate genes related to the aristolochic acid synthesis were screened,isolated and identified.This study provided an important scientific basis for studying the detoxification of Asarum heterotropoides Fr.Schmidt var.mandshuricum(Maxim.)Kitag at the molecular level.The main conclusions are as follows: 1.With analysis of gene differential expression and comparison with the KEGG database,it was found that there is 19 significant enrichment pathway involving 101,01 Unigenes,among which 97 Unigenes involved in the synthesis of isoquinoline alkaloids.Except for the sequence having repeated annotation information,9 Unigenes with aspartate aminotransferase activities were screened,and they were c290059_g1_i1,c211451_g1_i1,c199520_g1_i1,c211451_g1_i2,c215800_g2_i1,c149320_g1_i1,c377160_g1_i1,c191631_g2_i1,c174134_g2_i1.After further screening,c211451_g1_i2 was used as a candidate gene for aspartate aminotransferase,and this laid the foundation for further research.2.The expression of candidate gene c211451_g1_i2 was analyzed using the quantitative PCR technique,and use Actin as the internal reference gene.The results showed that the gene c211451_g1_i2 had higher expression in the leaves of Asarum heterotropoides Fr.Schmidt var.mandshuricum(Maxim.)Kitag 3.The gene c211451_g1_i2 was constructed into the prokaryotic expression vector and successfully expressed this recombinant protein in E.coli strain BL21.4.The recombinant plasmid for gene overexpression was successfully constructed,and successfully transferred the engineering bacteria into the callus of Asarum heterotropoides Fr.Schmidt var.mandshuricum(Maxim.)Kitag.