Molecular Cloning, Characterization And Functional Analysis Of Salt-Tolerance Genes From Brassica Napus

Some plant Na+/H+ genes have been successfully used in the genetic engineering to develop salt tolerance crops, which has been a hot spot of genetic engineering research. The full-length cDNA of Brassica napus antiporter gene(BnNHX1, GenBank Acc. No. AY189676) was cloned by RACE-PCR technique. Homology analysis and molecular modeling revealed that the BnNHX1 strongly resembled other Na+/H+ antiporter genes at the nucleic acid and amino acid levels. The deduced amino acid sequence of BnNHX1 had high similarity to AtNHX1 (87%), shared 71% similarity with amino acid sequence of Na+/H+ antiporter from Oryza sativa and was similar to Homo sapiens NHE (29%). The BnNHX1 had 10-12 transmembrane regions (M) at the amino-terminus. The transmembrane regions of BnNHX1 were found to share identities with those of other members of the Na+/H+ antiporter family and were highly conserved.In order to test BnNHX1's function in the plants, a plant expressing vector pCAMBIA2300- BnNHX1 for transforming dicot plants was constructed. The recombinant vector was subsequently introduced into Agrobacterium tumefaciens with freeze-thaw method. The leaf discs of tobacco plants were infected by A. tumefaciens and kanamycin-resistant plants were regenerated on the selection medium containing kanamycin. PCR and Southern blot analyses confirmed that the BnNHX1 gene was integrated into transgenic tobacco genome. Northern blot analysis showed that transgenic tobacco plants expressed BnNHX1 at various levels. Analysis for the T1 progenies derived from seven independent transgenic primary transformants expressing BnNHX1 showed that the transgenes in most tested independent T1 lines were inherited at Mendelian 3:1 segregation ratios. Transgenic T1 progenies could express BnNHX1 and had salt tolerance at levels comparable to their T0 parental lines. This study implicates that the BnNHX1 represents a promising candidate in the development of crops for enhanced salt tolerance by genetic engineering.In the meantime, the full-length cDNA of Brassica napus sos2 (Bnsos2, GenBank Acc. No.AY310413) was cloned by the RACE-PCR technique. The gene encoded a serine/threonine kinase. Homology analysis and molecular modeling revealed that Bnsos2 strongly resembled other sos2 genes at the nucleic acid and amino acid sequence level. The cloning of these genes provides a basis for the engineering of crop plants for enhanced salt tolerance in the future.