No abstract

An 808 bp promoter from a rice polyubiquitin gene, rubi3, has been isolated. The rubi3 gene contained an open reading frame of 1,140 bp encoding a pentameric polyubiquitin arranged as five tandem, head-to-tail repeats of 76 aa. The 1,140 bp 5' UTR intron of the gene enhanced its promoter activity in transient expression assays by 20-fold. Translational fusion of the GUS reporter gene to the coding sequence of the ubiquitin monomer enhanced GUS enzyme activity in transient expression assays by 4.3-fold over the construct containing the original rubi3 promoter (including the 5' UTR intron) construct. The enhancing effect residing in the ubiquitin monomer coding sequence has been narrowed down to the first 9 nt coding for the first three amino acid residues of the ubiquitin protein. Mutagenesis at the third nucleotide of this 9 nt sequence still maintains the enhancing effect, but leads to translation of the native GUS protein rather than a fusion protein. The resultant 5' regulatory sequence, consisting of the rubi3 promoter, 5' UTR exon and intron, and the mutated first 9 nt coding sequence, has an activity nearly 90-fold greater than the rubi3 promoter only (without the 5' UTR intron), and 2.2-fold greater than the maize Ubi1 gene promoter (including its 5' UTR intron). The newly created expression vector is expected to enhance transgene expression in monocot plants. Considering the high conservation of the polyubiquitin gene structure in higher plants, the observed enhancement in gene expression may apply to 5' regulatory sequences of other plant polyubiquitin genes.

Introns play a very important role in regulating gene expression in eukaryotes. In plants, many introns enhance gene expression, and the effect of intron-mediated enhancement (IME) of gene expression is reportedly often more profound in monocots than in dicots. To further gain insight of IME in monocot plants, we quantitatively dissected the effect of the 5' UTR intron of the rice rubi3 gene at various gene expression levels in stably transformed suspension cell lines. The intron enhanced the GUS reporter gene activity in these lines by about 29-fold. Nuclear run-on experiments demonstrated a nearly twofold enhancement by the 5' UTR intron at the transcriptional level. RNA analysis by RealTime quantitative RT-PCR assays indicated the intron enhanced the steady state RNA level of the GUS reporter gene by nearly 20-fold, implying a strong role of the intron in RNA processing and/or export. The results also implicated a moderate role of the intron in enhancement at the translational level ( approximately 45%). Moreover, results from a transient assay experiment using a shortened exon 1 sequence revealed an important role of exon 1 of rubi3 in gene expression. It may also hint a divergence in IME mechanisms between plant and animal cells. These results demonstrated transcriptional enhancement by a plant intron, but suggested that post-transcriptional event(s) be the major source of IME.

Maize is an important food and feed crop in many countries. It is also one of the most important target crops for the application of biotechnology. Currently, there are more biotech traits available on the market in maize than in any other crop. Generation of transgenic events is a crucial step in the development of biotech traits. For commercial applications, a high throughput transformation system producing a large number of high quality events in an elite genetic background is highly desirable. There has been tremendous progress in Agrobacterium-mediated maize transformation since the publication of the Ishida et al. (1996) paper and the technology has been widely adopted for transgenic event production by many labs around the world. We will review general efforts in establishing efficient maize transformation technologies useful for transgenic event production in trait research and development. The review will also discuss transformation systems used for generating commercial maize trait events currently on the market. As the number of traits is increasing steadily and two or more modes of action are used to control key pests, new tools are needed to efficiently transform vectors containing multiple trait genes. We will review general guidelines for assembling binary vectors for commercial transformation. Approaches to increase transformation efficiency and gene expression of large gene stack vectors will be discussed. Finally, recent studies of targeted genome modification and transgene insertion using different site-directed nuclease technologies will be reviewed.

The development of wheat (Triticum aestivum L.) cultivars that are resistant to Wheat streak mosaic virus (WSMV), yet competitive in yield under nondiseased conditions, is an objective for breeding programs in the Great Plains. This field study was conducted to compare classical and transgenic sources of resistance to WSMV. Three sets of germplasm were evaluated. These included adapted cultivars with various levels of tolerance, transgenic wheat lines containing viral coat protein or replicase sequences from WSMV that showed resistance in greenhouse trials, and germplasm with resistance to WSMV due to a translocated segment of chromosome 4Ai-2 from Thinopyrum intermedium (Host) Barkworth and Dewey containing Wsm1. A replicated field trial was conducted at Bozeman, MT, over a two-year period to evaluate the effectiveness of these different sources of resistance to mechanical inoculation of WSMV. Adapted cultivars differed in their ability to tolerate WSMV with mean reductions in yield over the two years ranging from 41 to 74%. Incorporation of the replicase or coat protein gene from WSMV did not provide field resistance to viral infection and in general, transgenic lines yielded less than their parent cultivar, 'Hi-Line'. Wheat-Thinopyrum lines positive for a DNA marker linked to the Wsm1 gene had significantly reduced yield losses ranging from 5 to 39% compared with yield losses of 57 to 88% in near isogenic lines not having the Wsm1 gene. Yield of lines with Wsm1 in the absence of disease ranged from 11 to 28% less than yield of lines without Wsm1. Our results suggest Wsm1 provides the best source of WSMV resistance but a yield penalty may exist because of the presence of the translocation.

Native strains of Pseudomonasfluorescens exhibited in vitro antibiosis towards isolates of races 1 and 4 ofFusarium oxysporum f.sp. cubense, the Panama wilt pathogen of banana. The seedlings of Musa balbisiana seedlings treated with P. fluorescens showed less severe wilting and internal discolouration due to F. oxysporum f.sp. cubense infection in greenhouse experiments. In addition to suppressing Panama wilt, bacterized seedlings of M. balbisiana also showed better root growth and enhanced plant height.

Introns are important sequence elements that modulate the expression of genes. Using the GUS reporter gene driven by the promoter of the rice (Oryza sativa L.) polyubiquitin rubi3 gene, we investigated the effects of the 5' UTR intron of the rubi3 gene and the 5' terminal 27 bp of the rubi3 coding sequence on gene expression in stably transformed rice plants. While the intron enhanced GUS gene expression, the 27-bp fused to the GUS coding sequence further augmented GUS expression level, with both varying among different tissues. The intron elevated GUS gene expression mainly at mRNA accumulation level, but also stimulated enhancement at translational level. The enhancement on mRNA accumulation, as determined by realtime quantitative RT-PCR, varied remarkably with tissue type. The augmentation by the intron at translational level also differed by tissue type, but to a lesser extent. On the other hand, the 27-bp fusion further boosted GUS protein yield without affecting mRNA accumulation level, indicating stimulation at translation level, which was also affected by tissue type. The research revealed substantial variation in the magnitudes of intron-mediated enhancement of gene expression (IME) among tissues in rice plants and the importance of using transgenic plants for IME studies.

Wheat (Triticum aestivum) plants were stably transformed with the coat protein (CP) gene of wheat streak mosaic virus (WSMV) by the biolistic method. Eleven independently transformed plant lines were obtained and five were analyzed for gene expression and resistance to WSMV. One line showed high resistance to inoculations of two WSMV strains. This line had milder symptoms and lower virus titer than control plants after inoculation. After infection, new growth did not show symptoms. The observed resistance was similar to the 'recovery' type resistance described previously using WSMV NIb transgene and in other systems. This line looked morphologically normal but had an unusually high transgene copy number (approximately 90 copies per 2C homozygous genome). Northern hybridization analysis indicated a high level of degraded CP mRNA expression. However, no coat protein expression was detected.