Molecular Cloning, Expression Analysis And Sub-Cellular Localization Of Multidrug And Toxic Compound Extrusion Transporter(MATE) Gene In Blueberry

Blueberry(Vaccinium corymbosum) is one of the most commercially significant berry crops. Blueberry fruit, which are rich in anthocyanins and other bioactive substances, have received much attention due to their benefits to human health. However, the component and content of anthocyanins was different in different varieties. Breeding new varieties that are rich in anthocyanins by biotechonology strategy is the goal for researchers. It is well known that anthocyanins are synthesized by a cytosolic multienzyme complex in the endoplasmic reticulum(ER), however stored in the central vacuole. Anthocyanins biosynthesis was studied thoroughly, but the mechanisms involved in the downstream steps of the pathway, such as transport and vacuolar accumulation, remain unclear. Multidrug and toxic compound extrusion(MATE) proteins are the most recently identified family of Multidrug transporters. Plant MATE transporters involved in transport of anthocyanins. Anthocyanins can be recognized by MATE transporters, then transported and finally accumulated inside the vacuole by the change of conformation. Mining related MATE-type anthocyanins transport genes and functional verification will fill a major gap in the complete picture of blueberry metabolic pathways, and also will help us to develop better nutritional blueberry cultivars. 8 Vc MATE genes with high gene abundance and expression level were cloned from highbush blueberry ‘Patroit’. Bioinformatics analysis and correlation analysis between expression pattern and the anthocyanins accumulation were conducted. Base on the result, subcellular localization were observed for the most likely anthocyanins transportation gene. The results are as follows:1. Anthocyanins accumulated mainly in the pericarp cell, content of anthocyanins increased with the fruit development. Various vesicles which transport anthocyanins were present in blueberry fruit by microstructure observation. Vesicles which migrate toward the vacuole coalesce each other to form lager vesicles. There are large amounts of anthocyanin vacuolar inclusions in the central vacuole of pericarp cell in mature berry. Pericarp cell of mature berry were be filled with vesicles All-Unigenes related to anthocyanin transport, such as GST, ABCC(MRP) and MATE, were found in blueberry fruit transcriptome by Blast.2. Thirty-three Unigenes annotated as MATE transporters were found in the blueberry fruit transcriptome. 20 Unigenes were up-regulated in exocarp, of which 19 Unigenes were up-regulated significantly, and 13 Unigenes were up-regulated in sarcocarp, of which 10 Unigenes were up-regulated significantly.3. Genome walking and RACE technology were used to clone 5′ end and 3′ end of the full length c DNA of Vc MATEs. These sequences were submitted to Gen Bank, and the registration number were KF875433, KF831422, KF875434, KF875435, KF875436, KF875438, KF875439 and KF875440 respectively.4. These Vc MATE proteins are composed of 477~517 residues, with molecμlar masses ~54 k Da, and theoretical isoelectric points from 5.35 to 8.41. All Vc MATE proteins were predicted to be localized to the plasma membrane without an N-terminal signal peptide. Transmembrane region prediction showed there were 10~12 putative transmembrane segments for Vc MATEs by TMHMM program, however there were 12 putative transmembrane segments for Vc MATEs, except 13 for Vc MATE5 by TMHMM program.5. Eight MATE proteins shared 32.1~84.4% identity, containing 5 short stretches containing conserved amino acids. Vc MATE2, Vc MATE3, Vc MATE5, Vc MATE7, Vc MATE8, and Vc MATE9 were more similar to the MATE-type flavonoid transporters. Expecially, Vc MATE2 contained all the TT12-specific residues.6. Phylogenetic analysis revealed the presence of five distinct clusters(clusters I, II, III, IV and V), implying that the five clusters might differ functionally in some respect. Vc MATE2, Vc MATE3, Vc MATE5, Vc MATE7, Vc MATE8, Vc MATE9, and some known MATE-type flavonoid transporters were grouped into one subclade,indicating that they might be involved in flavonoid transport. While Vc MATE1, Vc MATE4 and some toxin exporter grouped into another clade, indicating they may be involved in the transport of secondary metabolites, the detoxification of xenobiotics, or the export of toxic cations.7. Real-time quantitative PCR demonstrated that the expression profile of the eight Vc MATE genes varied spatially and temporally. The expression level of Vc MATE2 was the highest among the 8 genes. Vc MATE2 transcript was found most strongly expressed in anthocyanin-rich exocarp, and followed by root. During fruit developmental stages, expression was low in green fruit stage and then increased after fruit coloring. Vc MATE3, Vc MATE8 and Vc MATE9 followed a similar expression pattern with Vc MATE2. Vc MATE5 and Vc MATE7 present at higher transcript abundance in seed than exocarp. Transcript level of Vc MATE1 and Vc MATE4 were much weaker than other genes, and the highest expression site for the two genes was sarcocarp rather than exocarp.8. Correlation analysis between expression and anthocyanin accumulation indicated that there were some correlation between the expression profile and the accumulation of anthocyanins, the correlationship between expression of Vc MATE2, Vc MATE3, Vc MATE8 and Vc MATE9 and anthocyanin accumulation were significant at 0.01 level. In addition, expression level of Vc MATE2 was the highest among the 8 genes. So Vc MATE2 were regard as a predomainly anthocyanins transporter in blueberry.9. The result of Hmmunocytochemistry localization showed that Vc MATE2 was localized to the periphery of the pericarp cell.The Vc MATE2::YFP fusion gene was transformed into Arabidopsis protoplast by PEG mediated Ca Cl2 transformation, the results suggested that the Vc MATE2 protein was targeted to the tonoplast.