Cloning, Expression Analysis And Transformation Of The Vacuole Membrane Na～+/H～+ Antipoter Gene From Alfalfa

Vacuolar compartmentation of Na* through Na+/H+ antiporters in the large vacuole membrane is an essential mechanism for salinity tolerance of plants. First, sequestration of Na+ into vacuole could lower cytosolic Na+ level, which keeps redundant Na+ from the sites of metabolism and alleviates the toxic effects to enzymes and membrane system; Second, plant could take advantage of enough Na+ in vacuole to lower the water potential of cell and to resist the osmotic stress of salt. Moreover, efficient Na+ uptake into vacuole could maintain the high cytosolic K+/Na+ rate.By the multiply alignment of amino acid sequences and nucleotide acid sequences of several plants vacuole membrane Na+/H+ antiporters in Genbank, a pair of degenerate primers was designed against the conserved regions that was used for RT-PCR to amplify a 288bp cDNA segment from the medicago sativa L. Based on the sequence of the cDNA segment, primer was designed for 3' RACE and a 1536bp cDNA segment was acquired; from the sequence of 3' end of the gene, primer was designed for 5'RACE and a 1082bp cDNA segment was acquired. The full-length cDNA was gained by overlapping sequences, and the analysis of the sequence indicates that it containes an open reading frame, comprising 541 amino acid residues. The amino acid sequence compared by Blast revealed high homology with those of other plant vacuole membrane Na+/H+ antiporters, and the similarity to InNHX1 was the highest with 78%. The result indicates the gene cloned from alfalfa is a vacuole membrane Na+/H+ antiporter gene named MsNHX1, and its Genbank accession number is AY456096.Semi-quantitive RT-PCR was performed to reveal transcript level of MsNHXl in different tissues and under different abiotic stresses. The results indicated MsNHXl was abundant in leaf and stem, rather than in root; Furthermore, under the 200mM NaCl stress, the transcript level of MsNHXl was up-regulated and reached its peak after 6 hours, when drought and cold treatment could not.The coding region of MsNHXl was inserted into the expression vector PBI121 and the recombined vector was named as PBINHX. Then PBINHX was introduced into Agrobacterium tumefaciens LBA4404. Alfalfa was transferred by leaf disk, expecting to gain MsNHX1 overexpression plant. After selection by antibiotics, the regenerated plants were obtained and PCR analysis of them showed they were all PCR positive lines.