A dehydrin,dhn4, cDNA fragment has been obtained via RT-PCR from Tibetan hulless barley(Hordeum vulgereL. var. nudum Hook. f.). It indicated that dhn4encoded a YSK2 type dehydrin (DHN4). One Y segment (VDEYGNP), one S segment (SGSSSSSSS) and two K segments (RKKGIKEKIKEKLPG and EKKGIMDKIKEKLPG) were identified in the deduced amino acid sequence of dhn4.
The secondary structure of DHN4 protein predicated with software Anthepro 5.0 is prone to ?-helix, and the tertiary structure predicated by SWISS-PORT indicated intrinsically unstructured. The coding region of the dhn4 cloned into pBI121 binary vector with the 35S promoter was transformed into the Agrobacterium tumefaciens strain DHA105.
The Agrobacterium mediation was transformed dhn4 into the leaf disc of tobacco and then the tobacco plantlets with kanamycin resistant were regenerated using callus induction mediums supplemented with kanamycin and carbencillin. The regenerated plants were transferred into plots with peat moss and grown in the greenhouse. The inserting dhn4 of regenerated plants were identified separately by PCR, PCR southern blot and DNA sequencing using the gnomic DNA.