| Iron(Fe) deficiency is a worldwide spread serious problem in agricultural production. Iron deficiency problem is much more serious on alkaline soils. Graminaceous species secrete mugineic acids(MAs) into the rhizosphere for solubilizing and uptaking inorganic Fe(III). Only 2′-deoxymugineic acid(DMA) was secreted in Maize(Zea mays). We have cloned DMA secretion channel gene YS3. In this research, we studied YS3 gene expression pattern by analyszing the expression of GUS drived by the YS3 promoter, real-time quantitative PCR technology, immunohistochemical technology and subcellular localization technology, and laid the foundation for DMA secretion mechanism in molecular aspects. The results were summarized as follows:1. We cloned YS3 gene promoter pYS3. By prediction using promoter anayisis software, the promoter contain the conserved domains of TATA box and CAAT box, and a large numbers of cis-acting regulatory elements such as light response element, hormone( such as auxin, abscisic, MeJA) response element, circadian control regulatory element, stress response element, etc. Analysed the expression of GUS gene drived by pYS3, we found that the promoter could drive GUS gene expression mainly in root epidermal cells.2. Real-time qPCR was performed to determine the transcription levels of YS3 in different organs of maize, including leaf and root at earing stage from field, dry seeds of maturity, seeds after 48 h gramination, leaf and root of seeds living in water culture for two weeks under iron sufficient and iron deficiency conditions. The expression of YS3 gene was detected in almost all organs, but the expression in root was higher than in others. The YS3 gene in leaf and root was up-regulated under iron deficiency condition. For example the expression quantity of root increased 54 times. Iron deficiency enhanced YS3 gene expression.3. To determine the expression pattern of YS3 at cellular level, immunolocalization studies were performed using polyclonal antibodies against the specific peptides derived from YS3. YS3-GFP fusion gene was transiently expressed in onion epidermal cells. YS3-GFP fusion protein was observed with laser scanning confocal microscope to determine its expression pattern at cellular level. The results revealed that YS3 was localized to epidermal cells and outer cortex cells of root at the cellular level, to cell membrane at the subcellular level. |
