Identification Of The ATP-binding Cassette Transporter Family Genes In Boraginaceae And Research On Key Functional Domains Of LeMDR3

Boraginaceae plants,such as Arnebia euchroma and Lithospermum erythrorhizon,are traditional Chinese herbal medicine plants in our country.Shikonin and its derivatives,naphthoquinone compounds which are isolated from the red roots of boraginaceae have a wide range of anti-tumor activities,antibacterial effects and antiinflammatory effects.The biosynthesis pathway of shikonin is mainly composed of the mevalonate pathway and phenylpropanine pathway.Based on the long-term researches on the catalytic enzymes in these two pathways,most of the nodes in the pathway have been relatively clear.Recently,some works have been published,making its biosynthetic pathway more and more clear.In addition to the biosynthetic process,the accumulation and transportation of intermediate products and final products during the synthesis process should also be considered in the study of secondary metabolites.In view of the fact that ABC transporters have been shown to be involved in material transport in many studies,it is of great scientific significance to identify members of the ABC transporter family from Boraginaceae based on whole-genome sequencing data.Moreover,in the previous work of our research group,an ABC transporter that might participate in the outward transport of shikonin was identified through the yeast heterologous functional complementation system,but its key functional domains need to be further clarified.In this study,the genomes of Lithospermum erythrorhizon and Echium plantagineum sequenced by our research group were used as bioinformatic analysis data and the callus cells of L.erythrorhizon were used as the main experimental materials.The ABC transporter family genes were identified and the expression patterns of important subfamily members in L.erythrorhizon were verified.Le MDR3 transporter gene was highly expressed during the synthesis of shikonin.Further study has clarified its key functional domains through the yeast heterologous expression system.At the same time,the combined analysis of transcriptome sequencing-mi RNA sequencing-degradome sequencing in L.erythrorhizon callus cells was used to find mi RNAs that may target and regulate the ABC transporter genes of L.erythrorhizon,providing ideas and basis for follow-up research.The main findings are as follow:(1)Using the ABC transporter genes of Arabidopsis thaliana as target sequences,a total of 126 homologous sequences were blasted from the L.erythrorhizon genome and 118 homologous sequences were blasted from the E.plantagineum genome.The analysis of conserved domains in ABC transporters indicated that the selected candidate genes were ABC transporter genes,and the basic information of these gene members were characterized.In view of the function of the whole-molecule ABC transporters in material transportation,the MDR,MRP and PDR transporter genes of L.erythrorhizon have been further analyzed,including the physicochemical properties,gene structures and sequence conservative motifs of amino acid in each subfamily member.(2)In the previous research,a yeast heterologous expression system was used to screen out an ABC transporter gene Le MDR3 that might be involved in the transport of shikonin.Since MDR transporters have been proved to be involved in the transportation of plant secondary metabolites in many studies,the expression patterns of 14 members of the MDR transporters in L.erythrorhizon have been analyzed.The results showed that Le MDR3 was significantly highly expressed during the production of shikonin.Through the structural analysis of the Le MDR3 protein sequence,point mutation technology was used to identify its key functional domains,which further explained that Le MDR3 may play an important role in the efflux process of shikonin.(3)In order to further analyze the expression regulating mechanism of the L.erythrorhizon ABC transporter genes,we used the combined analysis method of transcriptome sequencing-mi RNA sequencing-degradome sequencing to uncover 7mi RNAs possibly targetting the ABC transporter genes,which mi R399 a may target Le MDR1,mi R169 i may target Le MRP1 and mi R2603 may target Le PDR9.The results of this study not only provide an important reference for the in-depth study of the transport of intermediate products and final secondary metabolites of the Boraginaceae plants but also give an important guidance in the subsequent analysis of the expression regulating mechanism of the ABC transporter genes.