Cloning And Funcational Analysis Of ZIP Transporters In Blueberry

Blueberry(Vaccinium spp.)has strict requirements on soil quality,and is susceptible to iron(Fe)deficiency.To alleviate this problem,soil acification is applied to activate the rhizospheric Fe.However,rhizospheric acification always parrellels the activations of the heavy metals in soil and results in toxic effects.At present,the mechanisms of metal ion absorption and transport in blueberries are not clear.The ZIP transporter is capable of transporting Zn,Fe,Mn,Cu,Cd,Ni,Co and other metal elements,which plays an important role in the absorption of metal elements in plant roots,long-distance transport and steady-state maintenance of the above-ground,but little research has been reported on ZIP transporters in blueberries.Based on the analysis of the blueberry transcriptome database,this study isolated full-length cDNA of the ZIP transporter family genes from blueberry and the bioinformatics analysis was carried out.Then the functions of ZIP transporters in blueberry were proved by yeast functional complementation and further identified through transgenic Arabidopsis.Meanwhile,the physiological responses to different metal element treatments were assayed by hydroponic experiment,in order to reveal the biological function of blueberry ZIP transporter and further elucidate the mechanism of absorption and transport of metal elements in blueberry,which is beneficial to improve the inefficiency of mineral absorption of blueberry and alleviate the stress of heavy metal pollution on blueberry.The specific research results are as follows:Six blueberry ZIP family genes were cloned and named as VcZIP1,VcZIP2,VcZIP6,VcZIP7,VcZIP8,and VcZIP11,and the sequence length ranged 750-1200 bp,with th similarity as 20%-60%.In the phylogenetic tree,six genes fall into three relatively independent categories,indicating that they have different functions in plants.Transmembrane domain analysis showed that the metal ion binding region of the other five genes located in the middle of the transmembrane domain except for the VcZIP11 metal ion binding region,which facilitates metal ion binding and protein function playing.The length of the amino acid sequence of VcZIP11 is also shorter than that of other ZIP transporters,speculating that the expression of VcZIP11 in plants may be low,and its binding to metal ions is also relatively unstable.Under hydroponic conditions,iron deficiency induced 85 and 5% decline of Fe contents respectively in blueberry leaves and roots.Except for VcZIP7 in the leaves,the expression levels of the six genes were significantly up-regulated,which was 1.4-18.0 times that of the control.It may be deduced that six genes may be involved in Fe response mechanism in blueberries.However,under the treatment of excessive Fe,there was no significant change in the Fe content in various parts of blueberry,which may be related to the nutritional inefficiency of Fe in blueberry.When treated with excessive Zn,the transcripts of VcZIP1 and VcZIP2 in the roots was down-regulated by 41% and 73%,while the expression of VcZIP6 was up-regulated by 130% in the roots,indicating that VcZIP6 may be a low-affinity Zn transporter in blueberry.Under the treatment of excessive Zn,the expression levels of VcZIP1 and VcZIP7 in blueberry leaves were up-regulated by 34% and 30%.It was suggested that the up-regulated expression of VcZIP1 and VcZIP7 may be to promote the transport of Zn from root to shoot.At low and high concentrations of Cd,the expression of each gene was up-regulated to varying degrees.overexpressed of the above genes may be related to the antagonism of plants against the toxic effects of Cd.The Arabidopsis thaliana genetic transformation experiment further proved that VcZIP6,VcZIP7 and VcZIP8 have the function of transporting Fe.In the absence of Fe,wild-type Arabidopsis and each transgenic Arabidopsis plants showed typical Fe deficiency symptoms such as chlorosis and dwarf seedlings.Both 50 μM and 100 μM Fe·EDTA treatments could restore the Fe deficiency symptoms of the mutant plants to some extent,but the ZIP-overexpressed plants of the three genes all grew better under the treatment of 50 μM Fe·EDTA.It indicated that excessive Fe will also inhibit the growth of plants to some extent.In addition,the supply of sufficient exogenous Fe can restore the phenotype of wild-type Arabidopsis,suggesting that there may be a low affinity Fe absorption and transport mechanism,but further experimental evidence is needed.