Cloning, Expression Of Two Ammonium Transporter Genes OsAMT1; 4 And OsAMT5 In Rice And Gene Genetic Transformation

It has an important role to increase the nitrogen utilization efficiency, by which toimprove the environment and promote the sustainable development in agriculture. It isfound that some ammonium transporter genes have potential function in impoving thenitrogen uptake and utilization in plant under N-deficient condition. In this study, theOsAMT1;4 and OsAMT5 were cloned based on their cDNA sequences released inGenBank database in NCBI website, the international bioinformatics center. During thecloning, the DNA of TP309, a high-N efficiency rice variety, was used to PCRamplification of the genes.1. Sequencing analysis showed that there were no differences between the clonedgenes from TP309 with the released sequences in NCBI. OsAMT1;4 and OsAMT5 have anopen reading frame 1497 bp and 1377 bp, and encoding 498 aa and 458 aa, respectively.OsAMT1;4 and OsAMT5 all contained eleven transmembrane domains. Phylogenetic treeanalysis indicated that OsAMT1;4 could be classified into AMT1 subfamily, and OsAMT5into AMT2 subfamily.2 Using DNA reconminant techniques, the open reading frame of OsAMT1;4 andOsAMT5 were fused into the yeast expression vector P426 with no flame shift. Theconstructs were then transformed the yeast mutant strain 31019b, lacking the ability ofNH₄⁺ uptake. The results indicated that OsAMT1;4 and OsAMT5 all could recovery theNH₄⁺ uptake of the mutant, suggesting that the proteins translated by above genes have thefunction of NH₄⁺ uptake or translocation in rice plants.3 The expression patterns of OsAMT1;4 and OsAMT5 were analyzed based onsemi-quantative RT-PCR. The results indicated that no OsAMT1;4 transcripts weredetected under the experimental condition, including the regimes of NH₄⁺ and NO₃^-concentrations, and regimes of time cources with different time length at low NH₄⁺concentrations. The expression of OsAMT5 was leaf specific and inducible by high NH₄⁺concentrations, showing a increase of the transcript levels with the enchancement of NH₄⁺concentrations. Meanwhile, the expression level was increased with the prolonging oftreatment times under low NH₄⁺ concentration. There were no transcripts of OsAMT5detected in roots.4 The PCR products of OsAMT1;4 and OsAMT5 were ligated with the T-vector pUCm-T. The constructs were then transformed DH5αand positive clones were selected.The plasmids were isolated and a series of techniques were performed. Finally, the binaryvectors pCAMBIA1305-OsAMT1;4 and pCAMBIA1305-OsAMT5 which separatelyfused the OsAMT1;4 and OsAMT5 were constructed. The constructs were transformedAgrobacteria tumeficiens strain LBA4404. The tobacco were transformed with theLBA4401 strains carrying the binary expression constructs which fused the OsAMT1;4 andOsAMTS. Some positive seedlins were generated for further molecular identification.5 Based on PCR technology, the putative full length and deleted fragments ofOsAMT1;4 promoter were cloned. The framents were then fused into the correspondingposition of binary expression vector pCAMBIA1305, respectively. By this way, theconstructs in which the reporter gene GUS was driven by the deleted promoter fragmentswere established. The constructs were then transformed the Agrobacterium tumeficiensLBA4404 which were next used to transform the tobacco. The transgenic tobacco plantswere being genetated for further molecular analysis on regulatory elements evoluation inthe future.Based on the above work, the gene funcitions of OsAMT1;4 and OsAMT5 could beelucidated. It will also provide the theoretical basis for creating new crop germplasms orvarieties with high-N efficiency by genetic engineering technology.