Cloning And Functional Characterization Of AaPDR12 Transpoter Gene In Artemisia Aannua

Artemisinin,isolated from Artemisia annua L,has attracted gobal interest due to its medicinal value in treating malaria.Currently,the worldwide supply of artemisinin comes mainly from the extraction of A.annua plants.It is urgent to obtain high-producing plants with a large improvement of artemisinin accumulation,because the content of artemisinin in A.annua plants is very low.Great efforts showed that the treatment with abscisic acid(ABA)could significantly increase the artemisinin content in A.annua.The molecular mechanism of artemisinin accumulation enhanced by ABA has become a new focus in the study of artemisinin metabolic engineering.Besides,ABA transporter plays a crucial role in the synthesis of plant secondary metabolites.However,no ABA transporter has been reported in A.annua.In this study,AaPDR12 transporter gene was cloned from A.annua.The results on sequence analysis,evolutionary and expression analysis,transgenic A.annua study and in vitro transporting experiment using yeast mutant demonstrate AaPDR12 is involved in ABA transport.Besides,the promoter region of artemisinin biosynthetic enzyme gene AaADHl was cloned and the gene expression pattern in A.annua was analyzed.The main results are as follows:(1)A candidate gene AaPDR12 was identified and colned from the glandular trichome transcriptome database of A.annua by performing a BLASTP analysis using an ABA transporter protein gene AtPDR12 as a query.The expression of AaPDR12 was high in root;low in stem,bud,flower and young leaf;and poor in old leaf.Subsequently,the AaPDR12 expression pattern in leaves at different developmental stages was analyzed.AaPDR12 expression level was the highest in the youngest leaf and decreased rapidly with the leaf aging.The GUS activity was only observed in the root,stem,young leaf and trichome,indicating that AaPDR12 was expressed in tissue-specific pattern.In addition,AaPDR12 was proved to be a plasma membrane-localized protein.(2)The content of artemisinin was significantly increased and the accumulation of ABA was also promoted in the AaPDR12-overexpressing transgenic A.annua plants.Comparing with the control lines,AaPDR12-overexpressing transgenic plants were more sensitive to exogenous ABA treatment and showed higher drought tolerance,but AaPDR12 RNAi transgenic plants was less sensitive and showed lower drought tolerance.(3)Yeast cells expressing AaPDDR12 showed more efficient ability of ABA uptake.The result strongly demonstrated that AaPDR12 was an ABA importer in yeast.(4)The promoter region of AaADHl was cloned and some putative cis-elements were presented in the promoter sequence.The recombinant vector pCAMBIA1391Z-proAaADH1-GUS was introduced into A.annua plants and GUS singals were only detected in the glandular secretory trichomes of young tissues in transgenic A.annua plants.The AaADHl promoter may be a good substitute for constitutive promoter to drive the efficient and specific expression of AaPDDR12 gene in A.annua.In conclusion,AaPDR12 transporter gene was cloned from A.annua and identified to be involved in ABA transport.Additionally,overexpression of AaPDR12 significantly increased both endogenous ABA and artemisinin contents in transgenic A.annua plants.