Identification and use of the sugarcane bacilliform virus enhancer in transgenic maize.

BACKGROUND: Transcriptional enhancers are able to increase transcription from heterologous promoters when placed upstream, downstream and in either orientation, relative to the promoter. Transcriptional enhancers have been used to enhance expression of specific promoters in transgenic plants and in activation tagging studies to help elucidate gene function. RESULTS: A transcriptional enhancer from the Sugarcane Bacilliform Virus - Ireng Maleng isolate (SCBV-IM) that can cause increased transcription when integrated into the the genome near maize genes has been identified. In transgenic maize, the SCBV-IM promoter was shown to be comparable in strength to the maize ubiquitin 1 promoter in young leaf and root tissues. The promoter was dissected to identify sequences that confer high activity in transient assays. Enhancer sequences were identified and shown to increase the activity of a heterologous truncated promoter. These enhancer sequences were shown to be more active when arrayed in 4 copy arrays than in 1 or 2 copy arrays. When the enhancer array was transformed into maize plants it caused an increase in accumulation of transcripts of genes near the site of integration in the genome. CONCLUSIONS: The SCBV-IM enhancer can activate transcription upstream or downstream of genes and in either orientation. It may be a useful tool to activate enhance from specific promoters or in activation tagging.

Sugarcane DIRIGENT and O-methyltransferase promoters confer stem-regulated gene expression in diverse monocots.

Transcription profiling analysis identified Saccharum hybrid DIRIGENT (SHDIR16) and Omicron-Methyltransferase (SHOMT), putative defense and fiber biosynthesis-related genes that are highly expressed in the stem of sugarcane, a major sucrose accumulator and biomass producer. Promoters (Pro) of these genes were isolated and fused to the beta-glucuronidase (GUS) reporter gene. Transient and stable transgene expression analyses showed that both Pro( DIR16 ):GUS and Pro( OMT ):GUS retain the expression characteristics of their respective endogenous genes in sugarcane and function in orthologous monocot species, including rice, maize and sorghum. Furthermore, both promoters conferred stem-regulated expression, which was further enhanced in the stem and induced in the leaf and root by salicylic acid, jasmonic acid and methyl jasmonate, key regulators of biotic and abiotic stresses. Pro( DIR16 ) and Pro( OMT ) will enable functional gene analysis in monocots, and will facilitate engineering monocots for improved carbon metabolism, enhanced stress tolerance and bioenergy production.

Development of an activation tagging system for maize.

Activation Tagging, distributing transcriptional enhancers throughout the genome to induce transcription of nearby genes, is a powerful tool for discovering the function of genes in plants. We have developed a transposable element system to distribute a novel activation tagging element throughout the genome of maize. The transposon system is built from the Enhancer/Suppressor (En/Spm) transposon system and uses an engineered seed color marker to show when the transposon excises. Both somatic and germinal excision events can be detected by the seed color. The activation tagging element is in a Spm-derived non-autonomous transposon and contains four copies of the Sugarcane Bacilliform Virus-enhancer (SCBV-enhancer) and the AAD1 selectable marker. We have demonstrated that the transposon can give rise to germinal excision events that can re-integrate into non-linked genomic locations. The transposon has remained active for three generations and events displaying high rates of germinal excision in the T2 generation have been identified. This system can generate large numbers of activation tagged maize lines that can be screened for agriculturally relevant phenotypes.

A comprehensive study of the use of a homologous promoter in antisense cotton lines exhibiting a high seed oleic acid phenotype.

As opposed to first-generation biotechnology products, such as pest-resistant crops and herbicide-resistant crops, second-generation products often utilize plant-derived, homologous or heterologous genes and/or promoters. In this study, we evaluated the ability of a promoter from a gene encoding a major storage protein in cottonseed to drive an antisense sequence of the cotton FAD2 gene to down-regulate the activity of Delta-12 desaturase enzyme in cottonseeds. The oleic acid level in the transgenic cottonseeds approximately doubled from the wild-type level of 15%, with a concomitant decrease in the level of linoleic acid. A more extensive study of one line revealed a higher degree of seed-to-seed variability in the transgenic phenotype. A thorough investigation was conducted to determine the impact of the use of a homologous promoter to drive a transgene on the activity of the endogenous promoter. The results showed that the use of the homologous alpha-globulin B promoter for transgenic purposes did not adversely affect the expression of alpha-globulin B storage protein in cottonseed. The results obtained in this investigation on the use of a homologous promoter and antisense technology will be useful in the design of strategies to alter biosynthetic pathways for nutritional quality improvements and for the production of heterologous proteins of commercial value in seeds.

Cotton alpha-globulin promoter: isolation and functional characterization in transgenic cotton, Arabidopsis, and tobacco.

Globulins are the most abundant seed storage proteins in cotton and, therefore, their regulatory sequences could potentially provide a good source of seed-specific promoters. We isolated the putative promoter region of cotton alpha-globulin B gene by gene walking using the primers designed from a cotton staged embryo cDNA clone. PCR amplified fragment of 1108 bp upstream sequences was fused to gusA gene in the binary vector pBI101.3 to create the test construct. This was used to study the expression pattern of the putative promoter region in transgenic cotton, Arabidopsis, and tobacco. Histochemical GUS analysis revealed that the promoter began to express during the torpedo stage of seed development in tobacco and Arabidopsis, and during cotyledon expansion stage in cotton. The activity quickly increased until embryo maturation in all three species. Fluorometric GUS analysis showed that the promoter expression started at 12 and 15 dpa in tobacco and cotton, respectively, and increased through seed maturation. The strength of the promoter expression, as reflected by average GUS activity in the seeds from primary transgenic plants, was vastly different amongst the three species tested. In Arabidopsis, the activity was 16.7% and in tobacco it was less than 1% of the levels detected in cotton seeds. In germinating seedlings of tobacco and Arabidopsis, GUS activity diminished until it was completely absent 10 days post imbibition. In addition, absence of detectable level of GUS expression in stem, leaf, root, pollen, and floral bud of transgenic cotton confirmed that the promoter is highly seed-specific. Analysis of GUS activity at individual seed level in cotton showed a gene dose effect reflecting their homozygous or hemizygous status. Our results show that this promoter is highly tissue-specific and it can be used to control transgene expression in dicot seeds.