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.
"As proof-of-principle experiments to evaluate the application of this modular vector system for multigene delivery, two transformation vectors with different expression units for different aims of genetic improvement were developed. Several genes, including Atriplex hortensis betaine aldehyde dehydrogenase (badh) involved in the accumulation of glycine betaine giving tolerance to salt stress , isoflavone synthase (ifs) from Medicago truncatula that a key enzyme in the flavonoids/isoflavonoids pathway , a phosphinothricin resistance gene (bar) and two reporter genes, gus and gfp, were chosen for gene stacking. "
[Show abstract][Hide abstract] 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 05/2011; 6(5):e19883. DOI:10.1371/journal.pone.0019883 · 3.23 Impact Factor
"mangrove , spinach, amaranth, barley and sorghum, are proven betaine accumulators and tolerate salt and drought stress partly through this mechanism, but other species like tobacco, tomato and rice are considered non-accumulators of GB (Ishitani et al. 1993; Rathinasabapathi et al. 1993; Shirasawa et al. 2006). Transformation of the Badh gene from bacterial and plant sources into betaine-deficient plant species has resulted in accumulation of GB in their system and consequent acquisition of tolerance to salt and drought stress (Liang et al. 1997; Mohanty et al. 2002). BADH synthesis is up-regulated several-fold in response to salt and drought stress in spinach, barley and sorghum leaves (Weretilnyk and Hanson 1990; Ishitani et al. 1995; Wood et al.1996). "
[Show abstract][Hide abstract] 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.
"Bradbury et al. (2008) reported that the OsBADH1 and OsBADH2 enzymes are substrate-specific; for example, OsBADH2 have higher specificity to betaine aldehyde, which is the intermediate substrate for the glycine betaine biosynthesis pathway, than OsBADH1 enzyme. Although OsBADH2 has higher affinity to betaine aldehyde than OsBADH1, OsBADH1 has been reported to exhibit significantly increased transcriptional level when exposed to salt and drought stresses (Niu et al. 2007; Ishitani et al. 1994; Liang et al. 1997), whereas no consistent relationship between OsBADH2 transcription level and salt treatment was observed (Fitzgerald et al. 2008). This evidence suggests that only the OsBADH1 gene is probably involved in protecting plants against these stresses. "
[Show abstract][Hide abstract] ABSTRACT: Glycine betaine has been reported as an osmoprotectant compound conferring tolerance to salinity and osmotic stresses in plants.
We previously found that the expression of betaine aldehyde dehydrogenase 1 gene (OsBADH1), encoding a key enzyme for glycine betaine biosynthesis pathway, showed close correlation with salt tolerance of rice. In
this study, the expression of the OsBADH1 gene in transgenic tobacco was investigated in response to salt stress using a transgenic approach. Transgenic tobacco plants
expressing the OsBADH1 gene were generated under the control of a promoter from the maize ubiquitin gene. Three homozygous lines of T2 progenies with single transgene insert were chosen for gene expression analysis. RT-PCR and western blot analysis results
indicated that the OsBADH1 gene was effectively expressed in transgenic tobacco leading to the accumulation of glycine betaine. Transgenic lines demonstrated
normal seed germination and morphology, and normal growth rates of seedlings under salt stress conditions. These results suggest
that the OsBADH1 gene could be an excellent candidate for producing plants with osmotic stress tolerance.
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