The Two Genes Encoding Starch-Branching Enzymes IIa and IIb Are Differentially Expressed in Barley

The sbeIIa and sbeIIb genes, encoding starch-branching enzyme (SBE) IIa and SBEIIb in barley (Hordeum vulgare L.), have been isolated. The 5′ portions of the two genes are strongly divergent, primarily due to the 2064-nucleotide-long intron 2 in sbeIIb. The sequence of this intron shows that it contains a retro-transposon-like element. Expression of sbeIIb but not sbeIIa was found to be endosperm specific. The temporal expression patterns for sbeIIa and sbeIIb were similar and peaked around 12 d after pollination. DNA gel-blot analysis demonstrated that sbeIIa and sbeIIb are both single-copy genes in the barley genome. By fluorescence in situ hybridization, the sbeIIa and sbeIIb genes were mapped to chromosomes 2 and 5, respectively. The cDNA clones for SBEIIa and SBEIIb were isolated and sequenced. The amino acid sequences of SBEIIa and SBEIIb were almost 80% identical. The major structural difference between the two enzymes was the presence of a 94-amino acid N-terminal extension in the SBEIIb precursor. The (β/α)₈-barrel topology of the α-amylase superfamily and the catalytic residues implicated in branching enzymes are conserved in both barley enzymes.     

A Leader Intron of a Soybean Elongation Factor 1A (eEF1A) Gene Interacts with Proximal Promoter Elements to Regulate Gene Expression in Synthetic Promoters

Introns, especially the first intron in the 5’ untranslated region (5’UTR), can significantly impact gene expression via intron-mediated enhancement (IME). In this study, we demonstrate the leader intron of a soybean elongation factor 1A (eEF1A) gene (GmScreamM8) was essential for the high activity of the native promoter. Furthermore, the interaction of the GmScreamM8 leader intron with regulatory element sequences from several soybean eEF1A promoters was studied using synthetic promoters, which consisted of element tetramers upstream of a core promoter used to regulate a green fluorescent protein (gfp) reporter gene. Element tetramers, placed upstream of a GmScreamM8 core promoter, showed very high activity using both transient expression in lima bean cotyledons and stable expression in soybean hairy roots, only if the native leader intron was included, suggesting an interaction between intronic sequences and promoter elements. Partial deletions of the leader intron showed that a 222 bp intronic sequence significantly contributed to very high levels of GFP expression. Generation of synthetic intron variants with a monomeric or trimeric repeat of the 222 bp intronic sequence, yielded almost two-fold higher expression compared to the original intron, while partial deletion of the 222 bp intronic repeated sequence significantly decreased gene expression, indicating that this intronic sequence was essential for the intron-element interaction enhancement.     

Analysis of ZmAE3 upstream sequences in maize endosperm and androgenic embryos

The single copy gene ZmAE3 (androgenic embryo) has been isolated from early androgenic embryos of maize. It codes for a small hydrophilic protein with partial similarity to basal layer antifungal proteins. During normal reproductive development ZmAE3 is specifically expressed in the embryo-surrounding region, a specialised region of the endosperm. Here we report the cloning, sequencing and functional analysis of upstream sequences of ZmAE3. The fusion of a 1.1 kb potential promoter fragment of ZmAE3 to a uidA reporter gene resulted in g-glucuronidase activity in transiently transformed androgenic embryos and in Black Mexican Sweet maize suspension cells. The AE box, an 18 bp sequence present in the ZmAE3 promoter and in other endosperm-specific promoters, was shown to bind proteins present specifically in nuclear extracts of the lower half of the maize kernel. The potential role of this and other cis elements in the binding of trans-acting factors will be discussed.     

The promoter of the asi gene directs expression in the maternal tissues of the seed in transgenic barley

The bifunctional -amylase/subtilisin inhibitor (BASI) is an abundant protein in barley seeds, proposed to play multiple and apparently diverse roles in regulation of starch hydrolysis and in seed defence against pathogens. In the Triticeae, the protein has evolved the ability to specifically inhibit the main group of -amylases expressed during germination of barley and encoded by the amy1 gene family found only in the Triticeae. The expression of the asi gene that encodes BASI has been reported to be controlled by the hormones abscisic acid (ABA) and gibberellic acid (GA). Despite many studies at the gene and protein level, the function of this gene in the plant remains unclear. In this study, the 5-flanking region (1033 bp, 1033-asi promoter) and the 3-flanking region (655 bp) of the asi gene were isolated and characterised. The 1033-asi promoter sequence showed homology to a number of ciselements that play a role in ABA and GA regulated expression of other genes. With a green fluorescent protein gene (gfp) as reporter, the 1033-asi promoter was studied for spatial, temporal and hormonal control of gene expression. The 1033-asi promoter and its deletions direct transient gfp expression in the pericarp and at low levels in mature aleurone cells, and this expression is not regulated by ABA or GA. In transgenic barley plants, the 1033-asi promoter directed tissue-specific expression of the gfp gene in developing grain and germinating grain but not in roots or leaves. In developing grain, expression of gfp was observed specifically in the pericarp, the vascular tissue, the nucellar projection cells and the endosperm transfer cells and the hormones ABA or GA did not regulate this expression. In mature germinating grain gfp expression was observed in the embryo but not in aleurone or starchy endosperm. However, GA induced gfp expression in the aleurone of mature imbibed seeds from which the embryo had been removed. Expression in maternal rather than endosperm tissues of the grain suggests that earlier widespread assumptions that the protein is expressed largely in the endosperm may have been largely based on analysis of mixed grain tissues. This novel pattern of expression suggests that both activities of the protein may be primarily involved in seed defence in the peripheral tissues of the seed.     

Promoters derived from banana bunchy top virus-associated components S1 and S2 drive transgene expression in both tobacco and banana

Potential promoter regions of the Banana bunchy top virus (BBTV)-associated DNA components S1 and S2 were fused to the #-glucuronidase reporter gene and assessed for activity in both tobacco (Nicotiana tabacum cv. Xanthi) and banana (Musa spp. cv. Bluggoe). Transient assays indicated that all the S1- and S2-derived promoters were active and had greater expression in tobacco than banana. In stably transformed tobacco and banana, the S1- and S2-derived promoters directed expression in root meristems and trichomes. The S1 promoter was also expressed in the vascular tissue of leaves and roots, while both the S1 and S2 promoters were active in tobacco leaf trichomes and pollen. In banana, expression was significantly enhanced by the inclusion of the maize polyubiquitin intron 3' to the promoter. Interestingly, there was some evidence to indicate that S1 promoter fragments containing part of the open reading frame at the 5' end of the promoter had enhanced activity, suggesting that promoter elements may not be confined to the non-coding region.     

Mini-transposon Tn5gfp constructs for differential tagging of microorganisms

Recently thegfp (green fluorescent protein) gene from the jellyfishAequoria victoria has been widely used as a reporter gene. In this study mini-transposons, named as mini-Tn5gfp, were constructed by subcloning thegfp gene into a transposon Tn5. To improve the expression level of thegfp gene, tandom array ofgfp gene was obtained. The constructs were successfully used in tagging target microorganisms by transposition. The level of GFP expression was found to be closely correlated with the copy number of the gfp transposed. These constructs will facillitate not only efficient tagging of whole organism but also genetic marking of target genes by transposition.     

Conifer genetic engineering: transgenic Pinus radiata (D. Don) and Picea abies (Karst) plants are resistant to the herbicide Buster

A biolistic transformation procedure was applied to co-transform embryogenic tissue of Pinus radiata and Picea abies with two plasmid DNAs. The first vector contained the bar gene, specifying resistance to the herbicide glufosinate, under the control of the maize ubiquitin promoter. This plasmid also contained the Pinus radiata germin cDNA sequence, in either sense or antisense orientation, driven by the ubiquitin promoter. The second vector contained both the nptII gene under control of the CaMV 35S promoter for selection of transgenic tissue on geneticin and the uidA reporter gene under control of the double CaMV 35 promoter. Polymerase chain reaction analysis of selected geneticin-resistant tissue showed that the transformation rates for the co-bombarded plasmid were high in both Pinus radiata (75%) and Picea abies (86%). A combination of phenotypic analysis and Northern hybridisation demonstrated that a number of the transgenic lines expressed all four transgenes. Regenerated plantlets from Pinus radiata and Picea abies transgenic lines were spray-tested with commercial rates of Buster (glufosinate at 0.5, 1.0, 2.0 and 4.0 kg active ingredient per hectare). Transgenic plants survived and continued to grow with minor or no damage to their needles, whereas non-transgenic plants regenerated from the same cell lines died within 8 weeks of spraying. To our knowledge, this is the first report on genetically engineered herbicide resistance in conifers, and the results demonstrate that this trait is a feasible option for plantation forestry.     

Construction and testing of an intron-containing luciferase reporter gene from<Emphasis Type="Italic">Renilla reniformis</Emphasis>

We describe a newRenilla reniformis luciferase reporter gene,RiLUC, which was designed to allow detection of luciferase activity in studies involvingAgrobacterium-based transient expression studies. TheRLUC gene was altered to contain a modified intron from the castor bean catalase gene while maintaining consensus eukaryotic splicing sites recognized by the plant spliceosome.RLUC andRiLUC reporter genes were fused to the synthetic plant SUPER promoter. Luciferase activity within agrobacteria containing the SUPER-RLUC construct increased during growth in culture. In contrast, agrobacteria harboring the SUPER-RiLUC gene fusion showed no detectable luciferase activity. Agrobacteria containing these gene fusions were cotransformed with a compatible normalization plasmid containing a cauliflower mosaic virus 35S promoter (CaMV) joined to the firefly luciferase coding region (FiLUC) and infused into tobacco leaf tissues through stomatal openings. The kinetics of luciferase production from theRLUC orRiLUC reporters were consistent, with expression of theRiLUC gene being limited to transiently transformed plant cells.RiLUC activity from the reporter gene fusions was measured transiently and within stably transformed tobacco leaf tissues. Analysis of stably transformed tobacco plants harboring either reporter gene fusion showed that the intron altered neither the levels of luciferase activity nor tissue-specific expression patterns driven by the SUPER promoter. These results demonstrate that theRiLUC reporter gene can be used to monitor luciferase expression in transient and stable transformation experiments without interference from contaminating agrobacteria.