Functional Study And Promoter Anlalysis Of Zinc/Iron-Regulated Transporter-like Protein PtZIP1,PtZIP6 And PtIRT1 Of Trifoliate Orange(Poncirus Trifoliata L. Raf.)

Fe is a necessary mineral nutrition for plant growth and development.It participates in many physiological and biochemical processes,such as plant metabolism,protein synthesis and so on.Grafted plants such as citrus absorb Fe nutrition mainly through underground rootstocks.In citrus production,trifoliate orange(Poncirus trifoliata L.Raf.)is the most widely used rootstock because of its cold tolerance and resistance to root rot.However,one of big disadvantage of trifoliate orange is sensitive to alkaline soil.Citrus trees grafted on trifoliate orange often perform seriously deficient in Fe on alkaline soil,which resulted in weak of the tree and decline of yield and quality.It is difficult to correct by fertilizer application or other measures.ZIP family proteins are thought to be important transporters of many bivalent metal ions,such as Zn,Fe,Mn and Cu,which are involved in the regulation of Fe absorption and homeostasis in plants.Previously,12 members of the ZIP family were cloned from trifoliate orange in our lab.To further uncover their function,three candidate members,PtZIP1,PtZIP6 and PtIRT1,which may be involved in Fe absorption,were selected for functional study.This study can help to establish the foundation for creating Fe tolerant citrus rootstocks via genetic improvement.In this study,the expression patterns of PtZIP1,PtZIP6 and PtIRT1 under Fe and Zn deficiency were analyzed,the promoters were cloned and the promoter components were analyzed,and the subcellular localization was analyzed by transient expression of tobacco.Verification of ion absorption ability of yeast by complementation of yeast mutant with Fe,Zn,Mn absorption defect.In addition,its function was verified by the genetic transformation of Arabidopsis and trifoliate.A detailed analysis of the transgenic Arabidopsis was performed.The main findings are as follows:1.Differences in spatio-temporal expression patterns of PtZIP1,PtZIP6,and PtIRT1 were observed in the absence of Fe and Zn deficiency in trifoliate orange.In general,the expression of tested genes were significantly induced in the roots than the leaves under Zn and Fe deficiency.Among which,PtZIP1 was dominant induced in Zn deficient roots and Fe deficient leaves,while PtZIP6 and PtIRT1 were mainly induced under Fe deficiency.2.The sequences length of the cloned PtZIP1,PtZIP6 and PtIRT1 promoter were 1111 bp,1507 bp,and 2457 bp,respectively.PtZIP1,PtZIP6,PtIRT1 promoter sequence and sweet orange corresponding promoter were 81.43 and 89.39%,64.27% homology,respectively.The predicted promoter elements of PtZIP1,PtZIP6 and PtIRT1 include promoter core elements and environmental response elements.Environmental response elements include light response element,stress response element and hormone response element.All of PtZIP1,PtZIP6 and PtIRT1 have a series of light response elements,such as Box4,BoxI-TCT-motif,etc.PtZIP1 have 5'UTR-Py-rich stretch promoter core element and TC-rich repeats stress element.PtZIP6 and PtIRT1 include TATA-box promoter core element and CAAT-box,CAT-box enhancer,as well as hormone response element Aux-RR Core,F-box,GARE-motif,and P-box etc.3.Subcellular localization of PtZIP6 and PtIRT1 by GFP fused transient expression in tobacco suggest that they are located on the cell membrane.4.Complementation analysis of PtZIP1,PtZIP6 and PtIRT1 in yeast mutants defective in the uptake of Fe(fet3fet4),Zn(zrt1zrt2),and Mn(smf1)showed that PtZIP1 can uptake Fe and Zn,PtZIP6 can uptake Fe,while PtIRT1 can absorb Fe,Zn and Mn simultaneously.5.Six PtZIP6-transformed Arabidopsis thanliana lines were obtained.Among them,the expression of PtZIP6 was high in Z6-1,Z6-2,Z6-5 and Z6-6 lines.Further analysis with these four overexpressed lines showed that there was obvious yellowing in wild type leaves under Fe deficiency,while only a few of leaves of transgenic plants showed slightly yellowing.The chlorophyll contents of transgenic lines were significant higher than the control.The Fe deficiency stress further induced the expression of PtZIP6 in transgenic lines,especially for Z6-2 and Z6-6.After recover Fe supply for 6 d,the chlorophyll content began to increase,especially for the Z6-2 and Z6-6 lines.After 3 days of Fe recovery,the expression levels of PtZIP6 significantly increased in all lines,.Expression analysis of endogenous AtZIP genes and the Fe-transporter related genes in transgenic lines showed that the expression of most genes were down-regulated in 4 transgenic lines under Fe deficiency stress,except up-regulation of AtPYE in Z6-1?Z6-2?Z6-5?Z6-6 lines.After recovery to normal nutrient solution,the expression of most of the endogenous genes were up-regualted,and the AtZIP1,AtZIP10,and AtIRT1 had highest expression levels in Z1-1?Z1-5?Z1-6 lines.6.Four PtZIP1 transformed transformed trifoliate orange lines were obtained in this study.qPCR detection suggests that the expression of PtZIP1 in transgenic lines were 1.3-6.0 times of those of WT control.