Cloning And Functional Analysis Of A Wheat MATE Transporter Gene

Multidrug And Toxic Compound Extrusion Family?MATE?is one of the five types of efflux transporter proteins and playS an important role in various organisms.In recent years,the study of MATE family proteins in Arabidopsis thaliana,a model plant,has also gradually increased.However,there is no study on this family of protein in wheat.In this study,a wheat MATE family transporter protein AK336065 was cloned and functionally studied and the following experimental results were obtained:?1?The phylogenetic tree of wheat,human,yeast,Arabidopsis MATE transporter family was constructed and these proteins share high homology.?2?By comparing the homologous sequences of AK336065 protein with the reported PfMATE protein,the results showed that there was a high homology between them.Based on the location of the 12 transmembrane domains of the PfMATE protein,the 12 transmembrane domains of the AK336065 protein were predicted.Through protein subcellular localization experiments,it was found that AK336065 protein overlaps many signals of the late endoplasmic localization protein ARA7,indicating that AK336065 protein is mainly localized in the late endosome.It is speculated that the AK336065 protein may be involved in cell-mediated biological processes.?3?The AK336065 overexpression transgenic line was obtained by constructing a binary vector and Agrobacterium-mediated genetic transformation.Overexpression of the AK336065 gene resulted in early flowering,increased leaf senescence and dwarfed clumping of rosette leaves in Arabidopsis.In the carbon starvation-induced plant senescence system experiment,it was found that AK336065 gene promotes leaf senescence.It shows that the AK336065 gene is involved in leaf senescence regulation and is a positive regulator of senescence.?4?It was found that the phenotype of the AK336065 overexpressing transgenic plant was similar to that of the abs3-1D mutant in laboratory studies.According to evolutionary analysis,the AK336065 belongs to the same evolutionary branch as ABS3,ABS4,ABS3L1,and ABS3L2,and it is guessed that their functions are similar with each other.By overexpressing the AK336065 gene in the mateq mutant,it was found that it can rescue the mateq mutant phenotype to the abs3-1D mutant phenotype.It showed that the AK336065 is functionally similar to the ABS3 and its homologous genes.?5?Wheat AK336065 protein may serve as an autophagy receptor with a conserved physical interaction with wheat ATG8 autophagic family proteins.Construction of BiFC vector through protoplast expression revealed a physical interaction between the AK336065protein and TaATG8d protein.The specificity of the interaction between these two proteins was verified by predicting the AK336065 protein AIM region,alanine substitution experiments,and bimolecular fluorescence complementation experiments.It was found that the interaction between AK336065 ^AIM1+AIM2 and TaATG8d protein was significantly inhibited after site-directed mutagenesis of the two predicted AIM1 and AIM2 in AK336065 protein.It was suggested that the AIM1 and AIM2 of AK336065 play an important role in the interaction with TaATG8d protein.