Functional Analysis Of Anthocyanin Transporters GSTF12 And TT12 In 'Red Zaosu' Pear

Anthocyanin synthesis and storage sites were separate,so the transport of anthocyanin to vacuoles was a very critical process.In this study,'Zaosu' pears and its red fruit peel sport ‘Red Zaosu'(Pyrus bretschneideri Rehd.)were used as the materials,and the genes PbGSTF12(Glutathione S-transferase F12)that encoding GST protein,PbTT12(Detoxification 35-like)that encoding MATE protein were screened out using receptacles and leaves transcriptome analysis.Through the gene expression analysis,transient injection experiment,Y1 H and Luciferase activity experiments studied the function of PbGSTF12 and PbTT12.These genes could transport and accumulate anthocyanin.The main findings are as follows:1.The genes PbGSTF12 and PbTT12 that encoding anthocyanin transporters were screened.In this study,we not only screened the genes related to anthocyanin synthesis from the transcriptome,such as the MYB transcription factors(Pb MYB10 and Pb MYB10b)and structural genes UFGT that have been reported in pears,but also the genes PbGSTF12 that encoding GST protein,PbTT12 that encoding MATE protein which were not reported in pear.2.Pb MYB10 and Pb MYB10 b would promote to accumulate anthocyanin by regulating the expression of PbGSTF12 gene in ‘Red Zaosu'.PbGSTF12 was highly homologous to glutathione S-transferase associated with anthocyanin transport in other species.The subcellular localization of PbGSTF12 protein was localized in the cytoplasm,cell membrane and nucleus.There was a high correlation between the expression level of PbGSTF12 gene and the content of anthocyanin(Correlation coefficient r = 0.91).Transient injection experiment also demonstrated when anthocyanin was synthesized,overexpression of PbGSTF12 would promote to accumulate more anthocyanin.The gene expression pattern in the transcriptome and q RT-PCR analysis showed that the expression level of Pb MYB10,Pb MYB10 b and PbGSTF12 were highly correlated in fruits and leaves,respectively.Therefore,it was preliminarily speculated that the PbGSTF12 may be regulated by the transcription factors Pb MYB10 and Pb MYB10 b.Y1H and Luciferase experiments verified that both Pb MYB10 and Pb MYB10 b could bind to the PbGSTF12 promoter and activate its expression.3.PbTT2(R2R3 MYB)would promote to accumulate anthocyanin by regulating the expression of PbTT12 gene in ‘Red Zaosu'.PbTT12 was homologous to the MATE2 which could transport anthocyanin in Arabidopsis thaliana.The subcellular localization of PbTT12 was localized in the cell membrane.The expression level of PbTT12 gene was significantly positive correlated with the anthocyanin content(r=0.65).The result of transient injection experiments also showed when anthocyanin was synthesized,overexpression of PbTT12 would promote to accumulate more anthocyanin.The uniprot online website preliminarily forecasted that PbTT12 would be regulated by the transcription factor PbTT2.The q RT-PCR results proved that PbTT2 and PbTT12 was correlated.The results of Y1 H and Luciferase activity experiments verified that PbTT2 could directly bind to the PbTT12 promoter and activate its expression.