Cloning And Analysis Of Rice OsNAT7 Gene Promoter

In previous research,our lab acquired a gene(OsNAT7) which has the highest ratio of -Fe/+Fe in rice roots based on the microarray analysis.To elucidate its regulatory mechanism at the transcriptional level,OsNAT7 promoter has been cloned and the sequence has been analysised by bioinformatic tools.Then the promoter activity in transgenic tobacco BY-2 cell suspension has been characterizatized in order to provide a insight into the role of OsNAT7 gene.Two putative cis-acting elements:IDE1-like and IDE2-like has been found in OsNAT7 promoter.To identify their function,1.5kb fragment of OsNAT7 promoter has been amplified by nest-PCR method,OsNAT7p::GUS has been constructed respectively and transferred into rice calluses by particle bombardment,successful expression of GUS gene was obtained.To identify the putative iron-deficiency responsive region,deletions from the 5'-end has been transformed into tobacco BY-2 cell suspension protoplast by PEG-mediated method.Transient expression revealed that OsNAT7 promoter diden't respond to iron-deficiency in tobacco BY-2 cell.To elucidate the expression pattern of OsNAT7 in tissue,plant expression vector pCam-OsNAT7p has been transformed into tobacoo,rice and tobacco BY-2 cell suspension by agrobacterium-mediated method.T₀ seedlings of transgenic tobaccos,transgenic rice calluses and transgenic BY-2 tobacco cell suspension was obtained.Inducible expression pattern of OsNAT7p::GUS in transgenic tobacco BY-2 cell suspension was analyzed.The results indicated that the promoter activity of OsNAT7 was not relative to iron\manganese\copper\zinc,but can be enhanced by citric acid.Citric acid can facilitate the uptake and transport of iron,little has known about the transcriptional regulation in response to citric acid.Further bioinformatic analysis combined with microarray results indicated that the expression of OsNAT7 gene was possiblely regulated by other gene which can respond to iron-deficiency.Plant hormone or physiological changs under iron-deficiency also have putative effects on the expression of OsNAT7 gene.