Function And Mechanism Of Multidrug And Toxic Compound Extrusion Protein MulMATE In Mulberry

Mulberry as the feed species of silkworm is the important material base of sericulture industry.In addition to being available for leaf gathering and silkworm rearing,mulberry also has high edible,medicinal,feeding and ecological values.Adversity is an important factor that restricts the geographical distribution of mulberry and affects the realization of economic and ecological value of mulberry.It is of great significance for the development of sericulture industry to explore the key genes in response to stress in mulberry and accelerate the selection and breeding of stress resistant varieties of mulberry.Multidrug and Toxic Compound Extrusion Family（MATE）proteins have a variety of biological functions and play an important role in plant growth,development and response to stress.However,there is no research report about MATE protein in mulberry.In this study,a mulberry MATE protein family gene was cloned and its subcellular localization,expression characteristics and biological functions were studied.Meanwhile,the interaction proteins of MulMATE protein were predicted and identified,and the mechanism of action of MulMATE protein was discussed.These results are expected to provide genetic resources for resistance breeding in mulberry,and also lay a foundation for the study of the function and mechanism of other members of the MATE protein family in mulberry.The main results are as follows:（1）Cloning and bioinformatics analysis of MulMATE gene.According to the transcriptome information,MulMATE gene was cloned and the encoding region of MulMATE contains 1440 bp,which encodes 479 amino acids and the theoretical molecular weight of the encoding protein is 52.553 k Da.It contains several phosphorylation sites,does not contain signal peptides and has 12 transmembrane domains.Multiple sequence homology analysis showed that the MulMATE protein was highly homologous to the MATE proteins of other species.The phylogenetic tree of MulMATE protein and Arabidopsis MATE gene family encoding proteins was constructed.The results showed that MulMATE protein had the closest genetic relationship with Arabidopsis DTX18 and DTX19 proteins.（2）MulMATE subcellular localization analysis.The subcellular localization analysis showed that MulMATE protein was localized to the cell membrane.After mutation of serine at position 4 of MulMATE into aspartic acid,MulMATE^S4D was still localized to the cell membrane and the signal intensity of subcellular localization did not change.However,when the 4th serine of MulMATE was replaced by alanine,the fluorescence signal intensity was significantly weakened or even lost under the same laser signal intensity and the localization of MulMATE^S4A in the cell membrane could not be identified,indicating that the phosphorylation of Ser4 may affect the stability and subcellular localization of MulMATE protein.（3）Expression characteristics and biological function of MulMATE gene.MulMATE gene was expressed in all tissues of mulberry without tissue expression specificity,but the expression abundance of MulMATE gene was different in different tissues and the expression abundance of MulMATE gene was higher in flowers and mulberries of mulberry.Overexpression of MulMATE gene in Arabidopsis thaliana resulted in smaller leaves,shorter plant type,shorter fruit pod length,decreased 1000-seed weight and shorter seedling hypocotyl,indicating that MulMATE gene was involved in regulating plant growth and development.In addition,the overexpression of MulMATE gene in Arabidopsis thaliana can increase the accumulation of anthocyanin in transgenic plants and proanthocyanidin in seed coat,suggesting that MulMATE gene may be involved in the regulation of anthocyanin and proanthocyanidin metabolism in plants.In addition,the overexpression of MulMATE gene in Arabidopsis thaliana can also enhance the tolerance of transgenic plants to aluminum,reduce the accumulation of aluminum ions in root tips,and enhance the tolerance of transgenic plants to Na Cl,exogenous ABA and PVP.Therefore,MulMATE gene also plays an important role in the response of plants to stress.（4）Prediction and identification of MulMATE interacting proteins.Through the bioinformatics website（String 11.0）,three kinds of proteins interacting with MulMATE were predicted:Mul5NG4,Mul WAT1 and Mul AECC8.The genes of three interacting proteins were cloned,and the interaction relationship between MulMATE and Mul5NG4,MulMATE and Mul WAT1,and MulMATE and Mul AECC8 was confirmed by double molecular fluorescence complementation assay,respectively.It was found that after site-directed mutagenesis of Ser4of MulMATE protein,the fluorescence signal of interaction between MulMATE^S4A and Mul5NG4/Mul WAT1/Mul AECC8 was weakened under the same laser intensity,while the interaction between MulMATE^S4D and Mul5NG4/Mul WAT1/Mul AECC8 was not changed,indicating that the phosphorylation of Ser4 of MulMATE protein affected the stability of the protein,thus affecting its interaction with the interacting protein and its function.