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Using RT-PCR method, the open reading frame (ORF) of AtNHX1-cDNA, encoding the vacuolar Na+/H+ antiportor, was cloned from Arabidopsis thaliana seedlings pretreated with 100 mmol/L NaCl for 24h. This ORF was inserted between CaMV35S promoter, a Omega fragment of TMV RNA 5'UTR and NOS polyA terminator in the T-DNA region of a binary expression vector pNT (Fig1). The recombinant plasmid, designated as pNT-AtNHX1, was then transformed into Agrobacterium tumefaciens LBA4404. Mediated by this engineering Agrobacterium, the AtNHX1 gene was transferred into T0 generation transgenic plant strains of A. melilotoides and 103 regenerated plants resistant to Kanamycin (Kan) were obtained. Some factors influencing the transformation efficiency, such as the concentration and infection duration of Agrobacterium, the concentration of Acetosyringone (AS), were optimized to establish a stable Agrobacterium mediated gene transformation protocol of A. melilotoides. PCR analysis, Southern blot and RT-PCR detection of some T0 transgenic plants showed that the AtNHX1 gene was evidently integrated into the genome of transgenic plants and couldl be transcripted properly. Under the same salt stress conditions, the detection of NaCl resistance revealed the difference between the wild-type calli and the transgenic calli that induced from the transgenic plants, i.e, the relative growth rates of the transgenic calli were remarkably higher than that of the wild-type calli. The K+ and Na+ contents and relative conductivity in the leaves of the transgenic plants and wild-type plants were estimated. It suggested that under the stress of different concentration of NaCl, K+/Na+ ratio in the transgenic plant cells were always higher than that in wild-type, however the situation of relative conductivity was on the opposite. From the facts above mentioned, the transformation of AtNHX1 gene not only enhanced the salt tolerance of transgenic A. melilotoides, but also reduced the cell membrane damage induced by salinity. |
