Cloning And Preliminary Inquiry Of A WRKY Transcription Factor AaWRKY23 In Anisodus Acutangulus

Anisodus acutangulus is a perennial herbs of Solanaceae and produce in Yunnan Province of China.The content of total alkaloids has up to 1.2% in A.acutangulus.It is higher compared with some other Solanaceae plants such as Hyoscyamus niger,Atropa belladonna and Datura Stramonium and it is a rare plant resource for developing and utilizing alkaloids.So,as a medicinal plant,it has important research and application value.The TAs are secondary metabolites from the Solanaceae plants including anisodamine,anisodine,hyoscyamine and scopolamine.Hyoscyamine and scopolamine are extensively used in clinical treament and the market demand is also very huge.Therefore,using genetic engineering to improve the production of hyoscyamine and scopolamine in A.acutangulus will effectively alleviate the contradiction between supply and demand of market tension.In this study,a novel WRKY gene,AaWRKY23 was cloned and isolated from A.acutangulus.We obtained the overexpression transgenic hairy root lines by genetic transformation and investigated the molecular characteristic and metabolish regulation work of AaWRKY23.The results are summarized as follows:(1)Eighty AaWRKY genes from A.acutangulus were identified by analysising transcriptome database.After removing the sequences which were repeated or containing incomplete WRKY structure domains,thirty-three AaWRKY genes were finally obtained.(2)Analysis of tissue expression profile of key enzyme genes in TAs synthesis pathway showed that AaPMT1,AaPMT2,AaTRI,AaCYP80F1 and AaH6 H all have highest expression level in root.(3)The tissue expression profile analysis of thirty-three AaWRKYs showed that AaWRKY1,2,4,5,7,8,9,11,12,13,14,15,17,19,21,23,25,27,28,29,30,31,32 have the highest expression level in root.These AaWRKY genes have the same tissue expression profiles as these key enzyme genes of TAs biosynthesis pathway.So we speculated that these AaWRKY genes may be involved in the synthesis of TAs.(4)These 23 AaWRKY genes which expressed highest level in root,the AtWRKYs in Arabidopsis,the NtWRKYs in Tobacco and some WRKY transcription factors that have been confirmed to participate in the regulation of plant secondarymetabolism were analyzed by phylogenetic tree and the results revealed that AaWRKY23 got together with CrWRKY1,OpWRKY1,AtWRKY70,AtWRKY54,SmWRKY70 ? 54.In addition,CrWRKY1 has been reported to participate in the regulation of secondary metabolism of plants.Therefore,we guessed that this WRKY transcription factor may be involved in the synthesis of TAs and then chosen this gene for further study.(5)We cloned and isolated the WRKY gene AaWRKY23 from A.acutangulus and conducted bioinformatics analysis of it,the results displayed that AaWRKY23 is mostly like to SlWRKY70.Sub-cellular localization results showed that this WRKY transcription factor fusion protein locates in the nucleus.(6)We obtained the overexpression transgentic hairy root lines of AaWRKY23 by C58C1 mediating genetic transformation of the stem segments of aseptic seeding in A.acutangulus.qRT-PCR analysis revealed that overexpressing AaWRKY23 promoted the expression of AaPMT1,AaPMT2,AaTRI,AaCYP80F1 and AaH6H.HPLC analysis suggested that TAs content was 1.946mg/g DW in the control lines,the average TAs content was 2.901mg/g DW in the AaWRKY23 overexpression lines.These conclusions showed that overexpressing AaWRKY23 can promote the accumulation of TAs.(7)The result of yeast one-hybridized experiment indicated that AaWRKY23 can interacted with the W-box of AaH6 H promoter,thereby regulating the expression of AaH6 H gene.(8)The Dual Luciferase experiment showed that AaWRKY23 can bind with the promoter of AaH6 H to promote its expression and the result consist with yeast onehybridized experiment.In summary: This paper we cloned a WRKY gene AaWRKY23 and studied its function in the synthesis of TAs.Its function and regulation mechanism in the synthesis of TAs in A.acutangulus were revealed.It provides a theoretical basis and experimental basis for improving the content of TAs in A.acutangulus by transcription factor strategy.