Identification Of Traditional Chinese Medicine Resources Of Andrographis Paniculata And Cloning And Expression Of WRKY TF Family Genes Related To Terpenoid Biosynthesis

Andrographis paniculata,also known as Chunlian Qiuliu,Yijianxi;Angiosperm,acanthaceae,Andrographis,annual herb,with a wide geographical distribution.A growing body of research has shown that it can inhibit atherosclerosis,treat high blood pressure,diabetes and cancer,and has been used to treat fever,colds,inflammation and other infectious diseases.Andrographis herbal medicines undergo traditional processing procedures such as broken and boiling before being sold in pharmacies;therefore,it is difficult to properly identify orrographis paniculata when processed into traditional Chinese medicine.The mini-barcode developed in this study is a good solution.Andrographolide is a very important metabolite of andrographis,and the WRKY transcription factor family is widely involved in the secondary metabolic pathway of Andrographolide.Based on the transcriptome data of Andrographis paniculata and bioinformatics,this study explores the important metabolic pathways of Terpenoid Biosynthesis.WRKY transcription factor gene,and its expression analysis and functional verification.The study obtained the following results:1.Development of a mini-barcode for the identification of Andrographis paniculataIn this study,17 samples of Andrographis paniculata and 29 processed samples were collected.Based on the ITS2 sequence,a nucleotide signature consisting of 110 bp was designed.This sequence is highly conserved and specific to Andrographis paniculata.Then,we used these new primers(XH1F/R)to amplify the nucleotide signature region from processed materials.We conclude that this 110 bp nucleotide signature could broaden the application of DNA barcoding to identify Chinese medicines and other products with degraded DNA.2.Analysis and verification of key WRKY transcription factor genes in the metabolic pathway of Andrographis paniculateIn this study,the extract of andrographolide was induced by methyl jasmonate.The WRKY transcription factor family genes up-regulated by MEJA treatment,andrographolide were identified by transcriptome sequencing and biosignal analysis.Four candidate genes,i.e.APWRKY9,APWRKY19,APWRKY36,APWRKY58 were cloned,and their spatial expression patterns were detected via qRT-PCR.It was confirmed that these four WRKY genes were differentially expressed among various tissues.These genes were separately overexpressed in tobacco.In both APWRKY19 and APWRKY36 transgenic tobacco plants,the expressions of terpenoid synthase genes NtCPS2 and NtABS were upregulated.The NtCPS2 gene in APWRKY19 transgenic tobacco has a significant up-regulation(P <0.05);the others have no significant changes.It is speculated that APWRKY19 may play a positive regulatory role in the synthesis pathway of plant mites.Through this result,it is expected to further reveal the molecular mechanism of WRKY transcription factor and purine synthesis pathway at the level of secondary metabolic regulation,and provide a theoretical basis for the clarification of the synthesis mechanism of the active constituents of terpenoids.