Cloning, Expression And Genetic Transformation Of A Novel Na～+/H～+ Antiporter Gene (Osant1) In Rice (Oryza Sativa)

The arable land is limited and being decreased presently in China. It has an important role to improve the crop productivity in the salty soil by which to promote the development of sustainable agriculture in China. The vacuole Na~+/H~+ antiporter, locating at the tonoplast, has a role to transport Na~+ from the cytoplasm to vacuole when the plants are met with more salt ions. By this way, the plants can maintain relative homeostasis in the cytoplasm and alleviate the damage from more Na~+. In this study, a novel rice (Oryza sitava) vacuole Na~+/H~+ antiporter gene, Osant1, showing high similarity with AtNHXl, was cloned from a rice variety (TP309) which shows high tolerance of Na~+. The gene structure, characterization of Osant1 and the expression pattern of Osant1were elucidated. Meanwhile, the binary vector of pCAMBIA1305-Osant1 and pCAMBIA1305-Osant1, fused the ORF of Osant1 and the promoter region of Osant1, were constructed. The constructed binary vectors were used to transform the model plant Arabidopdis based on the floral dipping transformation method mediated by Agrobacterium tumefeciens (Strain LBA4404). The transgenic plants fused Osant1 and Osant1 promoter were generated, which provide a platform for identification of Osant1 in salt tolerance capacity and exploration of Osant1 regulation mechanism in expression in future.The mainly results in this study are as follws:1. Based on the Arabidopsis vacuole Na~+/H~+ antiporter (AtNHX1) , a rice putative vacuole Na~+/H~+ antiporter gene (GenBank accession No: AK064004), showing high similarity with AtNHX1, was identified by BLAST analysis of AtNHX1 at the international bioinformatics website NCBI. The forward and reverse primer specific to the open reading frame (ORF) of AK064004 were used to PCR amplification of this novel Na~+/H~+ antiporter gene with the total RNA of leaves treated 150 mM NaCl. One about 1.6 Kb cDNA fragment was amplified and cloned in pUCm-T vector. The plasmid from potitive DH5a clone was sequenced and the insertion was 1608 bp.2. The sequence from the RT-PCR product was analyzed with AtNHXl based on the software DNAStar. It is found that the clone had high similarity with AtNHXl (73.3%) and was same as AK064004. It was named Osantl.The full length of Osantl cDNA is 2178 bp, includeing the ORF 1608 bp and 5' untranslated region of 179 bp and 3' untranslated region of 391 bp, respectively. At the genomic level, Osantl contains 14 introns and 15 exons.3. The translated protein of Osantl was characterized by protein analysis software ExPASy. Osantl encodes 535 amino acids. Osantl contains conserved motif LFFIYLLPPI usually found in vacuole Na+/H+ antiporter. The molecular weight of Osantl is 59.1 KD. Phylogenetic tree analysis indicated that Osantl has high similarities with the vacuole Na+/H+ antiporter from other plant species, such as Arab\dopsis(Arabidopsis thaliana), Maize(Zea mays), Whe&t(Triticum aestivum), Bailey(Hordeum vulgare), Iris lacteal, Phragmites australis and Thinopyrum elongatum. The transmembrane predication analysis founded that Osantl has 12 transmembrane domains. These results indicate that Osantl is a vacuole Na+/H+ antiporter.4. The Osantl transcripts in roots, stems and leaves treated 24 h of different NaCl concentrations including 0, 50 mM, 100 mM and 200 mM were analyzed based on semi-quantitative RT-PCR method. The transcripts in roots, stems and leaves were all low in the control plants (0 mM NaCl). The expression levels in roots, stems and leaves were increased with the increase of NaCl concentration in treatments. This indicated that the expression of Osantl was induced by NaCl.5. With the delay of NaCl treatment (200 mM NaCl) at treatment time point 0, 6 h, 24 h and 48 h, the transcripts of Osantl in roots, stems and leaves increased. Among the time points, compared to from 24 h to 48 h, the transcripts of Osantl from 0 h to 24 h had a more increase. This result indicated that the expression of Osantl under NaCl stress was regulated by the time of NaCl treatment.6. Based on routine molecular cloning techniques, the plant binary expression vector pCAMBlA\305-Osantl fused the ORF of Osantl with correct frame in pCAMBIA1305 was constructed. The binary vector pCAMBlA\305-Osantl was used to transform Agrobacterium Tumeficiens LBA4404. A positive LBA4404 clone was used to transform Arabidopsis by floral dipping method. The seeds containing transgenic plants were sterilized and sown on solidified MS medium containing 20 mg/L hygromycin and grown in a growth chamber. 5 transgenic plants were screened for further evaluation of salt tolerance.7. The promoter region locating at the 5' flanking region of Osantl was PCR amplified based on the sequence of AP004274 using TP309 DNA as template. The fragment length was 1385 bp. The PLACE software was used to identify the regulatory elements in the promoter. It is found that the Osantl promoter contains some important regulatory elements to control the expression of Osantl, including The CAAT box, functioning to recognize RNA polymerase and initiate gene transcription;the GATA box, which combine some transcription factor in rice;GT-1 box, which interacts transcription factors in GT-1 groups;the conserved TATA box, which regulates the transcription efficiency of downstream genes;and the CANNTG element, which can be frequently found in the promoters of genes induced by abiotic stresses. Based on molecular cloning techniques, the plant binary expression vector pCAMBIA1305-Osantl which fused Osantl promoter was constructed. The positive LBA4404 colony fused Osantl promoter was used to transform Arabidopsis by floral dipping method. The transgenic Arabidopsis plants were screened and generated for further study of the regulatory elements which regulate the Osantl expression. This result also provides a basis for fine identifying regulatory elements which control the gene expression under salt stress by promoter deletion analysis method.