Functional Identification Of Zinc Transporter In Zinc Nutrition Essential1(ZNE1) Of Arabidopsis Thaliana

Zinc is a very important microelement in animals and plants.When zinc is excess or deficient,it will affect the growth and health of animals and plants.Our team found that At3g08650,which encodes the zinc transporter in Arabidopsis.Under conditions of zinc excess,the growth of the two Arabidopsis mutants with T-DNA insertion of the gene were impaired compared to wild type.Their leaf veins were premature aging.They showed zinc excess sensitive phenotype.The above results indicated that At3g08650 may be involved in the uptake and accumulation of Zn²⁺in plant leaf cells,which plays an important role in the regulation of Zn²⁺homeostasis.Therefore,we named this gene ZNE1?ZINC NUTRITION ESSENTIAL1?.To investigate whether ZNE1 participates in the absorption of Zn²⁺and identify its functional sites.Firstly,we constructed fusion protein expression vectors by ZNE1gene and yeast expression vector pFL61 and transformed fusion vector into the zrt1zrt2?ZHY3?,which zinc uptake deficient.ZNE1 gene could make ZHY3 restore Zn²⁺uptake ability which indicated that ZNE1 has Zn²⁺transport function.Subsequently,we predict 10 possible phosphorylation sites by KinasePhos website which used as target of point mutation.AtZNE genes which contain S39A,W105R,G115R,S133A,G144D,S185A,G189V,V192L,S560I,T584A,double nucleotides?S185A,G189V?or triple nucleotides?S185A,G189V,V192L?mutations could not restores Zn²⁺uptake ability.This result confirmed that 10 phosphorylation sites are function sites involved in Zn²⁺transporting.Through analyzed protein structure by TMHMM website,ZNE1 has 14 transmembrane domains,8 short outside peptide l?named outside1-8?,7 short inside peptide?named inside1-7?.MicroCal i TC200 was used to determine the thermodynamic data of titration of different 12 short peptides with ZnSO₄.According to the titration curve,inside2 and outside1 were important short peptides which is Zn²⁺binding peptides.It is predicted that lysine and histidine often involved in Zn²⁺binding.According to yeast functional complementation experiments,It was found that lysine at position 126 and histidine at position 131 are Zn²⁺binding sites.The study of ZNE1 function is helpful to reveal the molecular mechanism of plant Zn homeostasis,and provide the theory for subsequent cultivation of optimized Zn nutrition crops.