Bioinformatics Analysis Of Ginseng MFSs And Transformation Of Carrots With Plastidic ATP/ADP Transporter Gene

The plastidic ATP/ADP transporter is located on the plastid membrane of cells and is responsible for the transportation of energy,which is an adenylate transport system in eukaryotes and has an effect on content of starch and lipid in plant.In this study,199 PgMFSs?Major Facilitator Superfamily,MFS?with complete conserved domain were screened from the constructed 248,993 unigenes database of Jilin ginseng using bioinformatics methods.We analyzed the gene ontology?GO?,expression analysis,evolutionary origin of the199 PgMFSs and so on.According to the bioinfomatics analysis results,the PgMFS060 was screened and transformed into carrots by Agrobacterium-mediated method to verify gene function.The main results of the study were as follows:1.The length of 199 PgMFSs were between 1000 and 3000 bp approximately.A total of 10GO sub-functions were annotated at level 2.The enrichment analysis showed that the five subregions of transport activity,membrane,cell part,organelle part,and membrane with a very significant difference compared to 248,993 unigenes databas.2.Phylogenetic tree was mapped using the NJ method,the result showed that 141 PgMFSs merging from different transcripts with complete conserved domains were divided into 8subfamilies.The same subfamily genes were clustered together nearly and most of them were conservative in evolution.3.We analyzed the expression patterns of PgMFSs in14 tissues of Jilin ginseng,42 farm cultivar and four different old-year roots,which showed tissue specificity and spatio-temporal specificity.The interaction patterns among genes were identified by constructing the interaction networks of genes,the results showed that most of the genes in the superfamily are co-transported into groups and participated in the expression regulation of life metabolism.4.PgMFS060 is one of the members of the MFS and is located on the plastid membrane of cells.It is responsible for energy transport and can participate in various physiological metabolic reactions in the plastids.5.The quantitative real-time RT-PCR results showed there is closely interaction relationship between PgMFS060 and the key enzyme genes involved in the saponin biosynthesis.The PgMFS060 expression level significant positively correlated with ginsenosides Rb₁,Rd and total saponin.6.The plant expression vector of PgMFS060 gene was constructed successfully and carrots were transformed by Agrobacterium-mediated method.Finally,24 positive transgenic plants were obtained,and the positive seedling rate was about 31.17%.The results of quantitative real-time RT-PCR showed that the gene was up-regulated in the tissues of roots,stems and leaves,the highest expression level was occurred in the leaves of positive seedling.The specific effects on the metabolites need to be detected later.7.Carrot callus suspension cultures were successfully domesticated,and the target gene in the callus of the Genetically modified carrot callus was stably inherited after multiple passages.