Structure and expression of the barley lipid transfer protein gene Ltp1

We have characterized a gene (Ltp1) encoding a barley lipid transfer protein. Northern blot analysis showed that Ltp1 mRNA accumulates specifically in the aleurone layer of developing and germinating seeds. Southern blot analysis indicated that LTP1 protein is encoded by a single gene in barley. Sequence analysis of Ltp1 showed that it contains an open reading frame of 351 bp interrupted by a single intron of 133 bp. Transient expression assays indicated that 702 bp of the 5 upstream region of Ltp1 is sufficient to direct aleurone-specific expression during late seed development and early germination.     

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

The bifunctional alpha-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 alpha-amylases expressed during germination of barley and encoded by the amyl 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.     

Expression of soybean lectin gene deletions in tobacco

A series of constructs containing the developmentally regulated soybean lectin gene (Le1) were used to transform tobacco plants in order to assess developmental and quantitative regulation conferred by flanking sequences. The largest of the lectin constructs contained approximately 3,000 base pairs (bp) of Le1 5 flanking region and 1,500 bp of the 3 flanking region. The smallest construct contained no 5 flanking region and 194 bp of the 3 flanking region. ELISA assays of lectin in individual tobacco seeds and Southern blot analyses confirmed that most constructs were inherited as unique insertion events. Maximal expression of Le1 required more than 338 bp of 5 sequence, indicating that far upstream factors are involved in quantitative control of lectin expression. Lectin expression declined more than 80% between deletions with 1,700 versus 338 bp of 5 flanking sequence. In contrast, developmental control of lectin expression was maintained by Le1 inserts with only 190 bp of 5 sequence. The lectin promoter offers a potential means to target high levels of gene expression to the developing seeds of soybean or other dicotyledonous plants.     

Promoter activity of the pER341-borne ST(Phsp) in heterologous gene expression in Escherichia coli and Streptococcus thermophilus(1).

A 138-bp EcoO109/HinfI fragment of Streptococcus thermophilus plasmid pER341 (2798 bp) including the promoter sequence of the heat stress protein gene hsp16.4 was tested in vector constructs for ability to activate the promoterless green fluorescent protein gene (gfp) from a jelly fish in Escherichia coli, S. thermophilus, and lactococci. ST(Phsp) promoted gfp expression in transformed hosts as evidenced by the presence of green fluorescent (GFP(+)) colonies under UV illumination. The results confirmed the potential of ST(Phsp) as a functional promoter in heterologous gene expression in dairy fermentation bacteria.     

Analysis of promoters in transgenic barley and wheat

Advances in the genetic transformation of cereals have improved the prospects of using biotechnology for plant improvement, and a toolbox of promoters with defined specificities would be a valuable resource in controlling the expression of transgenes in desired tissues for both plant improvement and molecular farming. A number of promoters have been isolated from the important cereals (wheat, barley, rice and maize), and these promoters have been tested mostly in homologous cereal systems and, to a lesser extent, in heterologous cereal systems. The use of these promoters across the important cereals would add value to the utility of each promoter. In addition, promoters with less sequence homology, but with similar specificities, will be crucial in avoiding homology-based gene silencing when expressing more than one transgene in the same tissue. We have tested wheat and barley promoters in transgenic barley and wheat to determine whether their specificity is shared across these two species. The barley bifunctional α-amylase/subtilisin inhibitor ( Isa ) promoter, specific to the pericarp in barley, failed to show any activity in wheat, whereas the wheat early-maturing ( Em ) promoter showed similar activity in wheat and barley. The wheat high-molecular-weight glutenin ( HMW-Glu ) and barley D-hordein ( D-Hor ) and B-hordein ( B-Hor ) storage protein promoters maintained endosperm-specific expression of green fluorescent protein (GFP) in wheat and barley, respectively. Using gfp , we have demonstrated that the Isa and Em promoters can be used as strong promoters to direct transgenes in specific tissues of barley and wheat grain. Differential promoter activity across cereals expands and adds value to a promoter toolbox for utility in plant biotechnology.     

Retina-specific expression from the IRBP promoter in transgenic mice is conferred by 212 bp of the 5'-flanking region

IRBP is a photoreceptor-specific glycoprotein that has been suggested as a retinoid carrier in the visual process. Previous research has shown that 1.3 kb of 5'-flanking sequence from the human IRBP gene is sufficient to promote photoreceptor-specific expression of reporter genes in transgenic mice. To define more narrowly the sequences that promote tissue-specific expression, chimeric constructs with shorter promoters were used to generate transgenic mice. The bacterial CAT gene was fused to fragments of 706 bp or 212 bp from the 5' end of the human IRBP gene. Analysis of the three transgenic families bearing the 706 bp IRBP promoter revealed that CAT expression was confined to the neuro-retina and the pineal gland. Analysis of the four transgenic families bearing the 212 bp IRBP promoter revealed the same tissue-specific CAT expression in three families. These results establish that tissue-specific expression of IRBP can be regulated by a short 212 bp promoter which has been conserved between humans and mice.