Cloning of HvBADH1 Gene from Hulless Barley and Its Transformation to Tobacco

Abstract

To reveal the relationship between the expression of betaine aldehyde dehydrogenase (BADH) and the tolerance to salt stress resistance in hulless barley ( Hordeum vulgare L. var . nudum Hook. f), a 1,512 bp cDNA encoding BADH was cloned from hulless barley using the methods of reverse transcription PCR (RT-PCR) and rapid application of cDNA ends (RACE). The cDNA, designated HvBADH1 under the accession number EF492983 in GenBank, encodes a 54.2 kD protein with 232 amino acid residues. Gene HvBADH1 exhibited a homology (98.4%) in amino acid sequence with BBD2 gene encoding an isoenzyme of BADH from barley. It also shared a high homology of 97.0%, 84.7%, and 85.1% with BADH in wheat ( Triticum aestivum L.), maize ( Zea mays L.), and rice ( Oryza sativa L.), respectively. Gene HvBADH1 was inserted into pMAL c2x, and the recombined plasmid was then transferred into Escherichia coli TB1 cells. The recombinant TB1 harboring pMAL c2x- HvBADH1 and the control TB1 harboring empty pMAL c2x cells were induced with IPTG. The results revealed that the recombinant E. coli cells expressed a fusion 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 open reading frame (ORF) of HvBADH1 was inserted between CaMV35S promoter and NOS polyA in T-DNA region of the binary expression vector pCAMBIA1301. The recombinant plasmid, designated pCAM-ba, was transferred into Agrobacterium tumefaciens strain LBA4404. The HvBADH1 gene was transformed to tobacco ( Nicotiana tabacum L,) mediated by Agrobacterium. Two hygromycin B (Hyg)-resistant plants were obtained. 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 was expressed on the mRNA level in the transgenic tobacco plants. This suggested that HvBADH1 gene was related to the salt tolerance in hulless barley, which can be expressed in transgenic plants.