Expression of the spinach betaine aldehyde dehydrogenase (BADH) gene in transgenic tobacco plants.
ABSTRACT Plasmid pLS9 contains a 1.5-kb of spinach cDNA including its complete open reading frame. The 1.5-kb BADH cDNA was cut from pLS9 using restriction enzyme and was inserted into the expression cassette of plasmid pYH between the CaMV 35S promoter and polyA signal sequence. The 35S-BADH cDNA-polyA fragment of pYH was cloned into a polylinker cloning site of the binary vector pBin19. The resulting plasmid pBinBADH-S was transferred to Agrobacterium tumefacies LBA4404. The tobacco plants were transformed with strain LBA4404 containing pBinBADH-S, and more than ninety kanamycin-resistant transformants were selected. Polymerase chain reaction (PCR) detection showed that more than 60% of the transformed tobacco plants contained the foreign BADH gene. The Western blot analysis, BADH enzymatic assay, specific stain for BADH activity, and the test for salt tolerance showed that BADH gene was normally expressed in the transgenic tobacco plants. The BADH enzymes also presented in chloroplasts and cytosol of the transgenic plants. The transgenic tobacco plants having strong expression of BADH gene had strong ability to tolerate high salt stress.
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ABSTRACT: The use of transgenes to improve complex traits in crops has challenged current genetic transformation technology for multigene transfer. Therefore, a multigene transformation strategy for use in plant molecular biology and plant genetic breeding is thus needed. Here we describe a versatile, ready-to-use multigene genetic transformation method, named the Recombination-assisted Multifunctional DNA Assembly Platform (RMDAP), which combines many of the useful features of existing plant transformation systems. This platform incorporates three widely-used recombination systems, namely, Gateway technology, in vivo Cre/loxP and recombineering into a highly efficient and reliable approach for gene assembly. RMDAP proposes a strategy for gene stacking and contains a wide range of flexible, modular vectors offering a series of functionally validated genetic elements to manipulate transgene overexpression or gene silencing involved in a metabolic pathway. In particular, the ability to construct a multigene marker-free vector is another attractive feature. The built-in flexibility of original vectors has greatly increased the expansibility and applicability of the system. A proof-of-principle experiment was confirmed by successfully transferring several heterologous genes into the plant genome. This platform is a ready-to-use toolbox for full exploitation of the potential for coordinate regulation of metabolic pathways and molecular breeding, and will eventually achieve the aim of what we call "one-stop breeding."PLoS ONE 01/2011; 6(5):e19883. · 3.73 Impact Factor
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ABSTRACT: Betaine aldehyde dehydrogenase (BADH) is a key enzyme involved in the synthesis of glycinebetaine—a powerful osmoprotectant against salt and drought stress in a large number of species. Rice is not known to accumulate glycinebetaine but it has two functional genes coding for the BADH enzyme. A non-functional allele of the BADH2 gene located on chromosome 8 is a major factor associated with rice aroma. However, similar information is not available regarding the BADH1 gene located on chromosome 4 despite the similar biochemical function of the two genes. Here we report on the discovery and validation of SNPs in the BADH1 gene by re-sequencing of diverse rice varieties differing in aroma and salt tolerance. There were 17 SNPs in introns with an average density of one per 171 bp, but only three SNPs in exons at a density of one per 505 bp. Each of the three exonic SNPs led to changes in amino acids with functional significance. Multiplex SNP assays were used for genotyping of 127 diverse rice varieties and landraces. In total 15 SNP haplotypes were identified but only four of these, corresponding to two protein haplotypes, were common, representing more than 85% of the cultivars. Determination of population structure using 54 random SNPs classified the varieties into two groups broadly corresponding to indica and japonica cultivar groups, aromatic varieties clustering with the japonica group. There was no association between salt tolerance and the common BADH1 haplotypes, but aromatic varieties showed specific association with a BADH1 protein haplotype (PH2) having lysine144 to asparagine144 and lysine345 to glutamine345 substitutions. Protein modeling and ligand docking studies show that these two substitutions lead to reduction in the substrate binding capacity of the BADH1 enzyme towards gamma-aminobutyraldehyde (GABald), which is a precursor of the major aroma compound 2-acetyl-1-pyrroline (2-AP). This association requires further validation in segregating populations for potential utilization in the rice breeding programs.Molecular Breeding 03/2010; · 3.25 Impact Factor
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ABSTRACT: Glycine betaine (GB) is a compatible quaternary amine that enables plants to tolerate abiotic stresses, including salt, drought and cold. In plants, GB is synthesized through two-step of successive oxidations from choline, catalyzed by choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH), respectively. Rice is considered as a typical non-GB accumulating species, although the entire genome sequencing revealed rice contains orthologs of both CMO and BADH. Several studies unraveled that rice has a functional BADH gene, but whether rice CMO gene (OsCMO) is functional or a pseudogene remains to be elucidated. In the present study, we report the functional characterization of rice CMO gene. The OsCMO gene was isolated from rice cv. Nipponbare (Oryza sativa L. ssp. japonica) using RT-PCR. Northern blot demonstrated the transcription of OsCMO is enhanced by salt stress. Transgenic tobacco plants overexpressing OsCMO results in increased GB content and elevated tolerance to salt stress. Immunoblotting analysis demonstrates that a functional OsCMO protein with correct size was present in transgenic tobacco but rarely accumulated in wild-type rice plants. Surprisingly, a large amount of truncated proteins derived from OsCMO was induced in the rice seedlings in response to salt stresses. This suggests that it is the lack of a functional OsCMO protein that presumably results in non-GB accumulation in the tested rice plant. KEY MESSAGE: Expression and transgenic studies demonstrate OsCMO is transcriptionally induced in response to salt stress and functions in increasing glycinebetaine accumulation and enhancing tolerance to salt stress. Immunoblotting analysis suggests that no accumulation of glycinebetaine in the Japonica rice plant presumably results from lack of a functional OsCMO protein.Plant Cell Reports 05/2012; 31(9):1625-35. · 2.94 Impact Factor