| In order to isolate ZIP genes from rice,the amino acid sequence of Arabidopsis ZIP transporter gene AtZIP1 was used as a query probe to search Rice Genome Database of China through tBLASTn algorithm program.Two cDNA fragments containing complete ORFs of 1,287bp and 1,207bp were respectively cloned from total RNAs prepared from rice seedlings by the RT-PCR approach,and designated as OsZIP7a(Accession No: AY275180) and OsZIP8(Accession No:AY324148).The predicted proteins OsZIP7a and OsZIP8 display high similarity to other plant ZIP proteins,with 384 and 390 amino acid residues in length respectively.Both of them contain 8 TM domains and a highly conserved ZIP signature motif,with a histidine-rich region in the variable sequence between TM domainsⅢandⅣin each protein.The semi-quantitative RT-PCR analysis showed that the expression profiles of OsZIP7a and OsZIP8 were constitutively and weakly expressed in roots,culms,leaves and flowering spikes at the adult stage.OsZIP7a was significantly induced by iron-deficiency only in rice roots.OsZIP8 was significantly induced by zinc-deficiency in both roots and shoots of rice seedlings.The fusion protein OsZIP7a::GFP was introduced to onion epidermal cells.The results by histochemical detection showed that the fusion protein was localized in the cytosolic membrane of onion epidermal cells,while the control of pBI121-GFP was distributed throughout the cells.The complementation analysis in yeast transformed with OsZIP7a and OsZIP8 were performed to determine whether OsZIP7a or OsZIP8 had Fe or Zn transporting capacity.When expressed in yeast(Saccharomyces cerevisiae) cells,OsZIP7a reversed the growth defects of the yeast iron-uptake mutant,and OsZIP8 reversed the growth defect of the yeast zinc-uptake mutant.It is suggested that these two proteins might function as iron or zinc transporters in rice respectively.The previous research showed that ZFP182 was up-regulated by cold,drought,high salinity and ABA treatment,ZFP245 up-regulated by cold and drought stress.In this study The fusion protein ZFP245::GFP was introduced to onion epidermal cells.The results showed that the fusion protein ZFP245::GFP was localized in the nucleus of onion epidermal cells,while the control of pBI121-GFP was distributed throughout the cells. About 1,980 bp sequences in the promoter region of the ZFP182 and ZFP245 were respectively fused to GUS::GFP reporter genes in the vector of pCAMBIA1304,and then transformed into rice by Agrobacterium-mediated transformation.Histochemical analysis revealed that the GUS activity of ZFP245 is more than of ZFP182 in the root,culm,leaf and seed of rice transgenic plants under the normal condition.GFP activities in the leaf and root of transgenic plants were increasely induced under 150 mM NaC1 or the cold(4℃) treatment.Transgenic rice plants of cultivars Zhonghua 11 and Nipponbare with overexpression or suppressexpression of both ZFP182 and ZFP245 were respectively generated by Agrobacterium-mediated transformation.There was no significantly difference in agronomic traits between 35S:ZFP182 and 35S:ZFP245 transgenic plants and vector transgenic or non-transgenic plants.Transgenic plants in the T2 generation were analyzed for abiotic stresses.The results showed that overexpression of ZFP182 and ZFP245 in rice conferred tolerance to salt,cold and drought stress.Microarray analysis was used to characterize the expression profiles of transgenic rice of over-expressing ZFP182 at transcriptional levels.There were 163 up-regulated genes and 243 down-regnlated genes. The possible functions of some genes regulated by ZFP182 transcriptional factor were analyzed through the BLAST(basic local alignment search tool) of NCBI(national center of biological information). |
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