Novel Determinants of Salinity Tolerance

摘要

A novel method for the isolation of plant cDNA clones that provide functional sufficiency for salinity tolerance in E. coli cells was developed. A cDNA library containing genes expressed in salt-adapted tobacco cells was constructed in the lambda ZAPII vector. E. coli cells were infected with the rescued pBluescript phagemid library, and the infected bacterial cells were selected on agar plates containing LB medium supplemented with a non-permissive concentration of NaCl, and 10 mM IPTG. After repeated selections, 10 colonies were chosen. These colonies contained plasmids with inserts from 0.8 to 2.0 kb in size. The inserts were isolated and introduced into several strains of E. coli. Cells transformed with plasmids containing the selected inserts exhibited tolerance to 1.17 M NaCl in LB medium in the presence of IPTG. Southern blot analysis using the respective cDNA inserts confirmed the presence of the DNA sequences in the tobacco genome. Northern blot analysis demonstrated greatly increased expression of the corresponding genes in salt-adapted tobacco cells. Expression of any of these cDNA sequences in E. coli was sufficient to provide enhanced tolerance to salinity. Nucleotide sequence analysis and a subsequent search for homology with other genes in the GenBank database resulted in the identification of a homolog of a MIP protein similar to aquaporins, a novel elongation factor eF-1 alpha , a CCAAT-box binding protein, and several novel genes not present in the gene bank. Functional sufficiency of plant cDNA clones in a prokaryotic model system provides a very powerful tool for the isolation of functionally important novel genes.