Cloning, Subcellular Localization Of Permease Gene And Analysis Of Genes Related To Vesicle Trafficking Response To Iron Deficiency In Rice

Iron is an essential micronutrient with numerous cellular functions, and plays a critical role in important biochemical processes. Iron deficiency can be particularly pronounced in plants grown on calcareous soils, which cover approximately one-third of the earth's surface. Iron deficiency is not only affective development of plants, if severe, can lead to reduction in crop yields and even complete crop failure, but also is an important reason for people's iron-deficiency.Rice, one of the most important cereal crop, is the food staple of more than half the world's population. It is an effective way to increase the yield of crop and solve the iron-deficiency problem by promoting the iron content in rice. With the completion of sequencing of the entire rice genome, the most effective strategy of studies on the expression of genes responded to Fe-deficiency in rice is functional genomic approaches. Microarray methods were employed through the use of a cDNA clones collection representing 10531 individual gene sequences for understanding deeply the strategy responded to lack iron stress. The highest ratio of -Fe/+Fe of transcripts relevant membrane vesicle were found among the whole up-regulated cDNAs. The vesicles in rice root apices were abundant and distinct, when the structural change of rice root apices under both Fe-deficiency and EDTA-Fe treatment observed with a transmission electron microscope, using the technique of ultrathin sections, which indicated that genes relevant to membrane vesicle may be expressed highly in Fe-deficiency.Microarray analysis was used to characterize the expression profile of rice at transcriptional levels in response to iron deficiency and FeC13 cultured in phytagel medium. There were total 309 distinguish cDNA spots, each gene among them was analyzed in the function through using the BLAST (basic local alignment search tool) of NCBI (national center of biological information).The analysis of the genes related to transporter (irt, nramp, ysl, Permease) or vesicle transporting and Ferritin gene by Real-Time PCR indicated that the transcription of irt, nramp, ysl, Permease all increase in iron deficient. Whereas the transcription of Ferritin increase under EDTA-Fe and FeCl₃ conditions.Permease gene with the full length of cDNA 1973bp has been cloned by RT-PCR method from iron-stressed rice root for 5days. Then two new plant expression vectors both sence and antisence pCAMBIA1302- Permease(S/A)-GFP were constructed respectively. Fused protein was transferred into onion epidermal cell by agrobacterium- mediated transformation or particle gun bombardment. Sub-cellular localization of permease protein was analyzed through GFP observed under scanning fluresent confocal microscopy. The results showed that permease protein was located in plasma membrane. Function .Hydropathy and transmembrane domain were analized through ConPredII softwear and NCBI and proved that permease span 12 transmembrane domain.It may may be tansport xanthine and uracil.Plant expression vectors had been transformed into tobacoo using leaf disc transformation , and obtained hygromycin-resistant buds in the MS medium and created rice suspension for researching cellular location in rice and transgenic rice.