关键词: 青稞, HvBADH1, 基因克隆, 遗传转化, 耐盐性, RT-PCR, RACE

Abstract: Glycine betaine is a nontoxic osmolyte accumulated in the cytoplasm of salt or drought stressed plants, marine animals, and microorganisms. Betaine aldehyde dehydrogenase (BADH) catalyzes the final step in the synthesis of glycine betaine from choline in many plants. In order to reveal the relationship between the BADH expression and salt stress resistance of hulless bar-ley (Hordeum vulgare L. var. nudum Hook.f.), a 1 512 bp cDNA encoding BADH was cloned from hulless barley using the methods of RT-PCR and RACE. This cDNA encoded a 54.2 kD protein containing 232 amino acid residues and HvBADH1 was designated with the accession number of EF492983 in GenBank. HvBADH1 exhibited the highest homology (98.4%) in amino acid sequence with BBD2 gene encoding an isoenzyme of BADH from barley. It also shared highly homology of 97.0%, 84.7%, and 85.1% with BADH wheat, maize, and rice respectively. The HvBADH1 gene was inserted into pMAL c2x and transformed into E.coli cells (TB1) for expression. The recombinant TB1 (harboring pMAL c2x-HvBADH1) cells and control TB1 (harboring empty pMAL c2x) cells were then induced with IPTG. The results revealed that the recombinant E. coli cells could express a fu-sion protein with molecular weight of 96.3 kD. This fusion protein was fused by maltose binding protein (MBP, about 42.1 kD) and the peptide (about 54.2 kD) encoded by HvBADH1. The ORF of HvBADH1 was inserted between CaMV 35S promoter and NOS polyA in T-DNA region of binary expression vector pCAMBIA1301. The recombinant plasmid, designated as pCAM-ba, was transformed into Agrobacterium tumefaciens LBA4404. The HvBADH1 gene was transformed to tobacco mediated by Agro-bacterium. Two hygromycin B (Hyg) resistant regenerated plant strains were selected. PCR detection and Southern blot analysis indicated that all the Hyg resistant tobacco plants contained the alien BADH gene. RT-PCR analysis showed that HvBADH1 gene normally expressed on the mRNA level in the transgenic tobacco plants. The results suggest that HvBADH1 gene is related with the salt tolerance in hulless barley and can express in transgenic plants.

Key words: Hordeum vulgare L. var. nudum Hook.f., HvBADH1, Gene clone, Genetic transformation, Salt-tolerant resistance, RT-PCR, RACE