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Gene silencing is an important but little understood regulatory mechanism in plants. Here we report that a viral sequence, initially identified as a mediator of synergistic viral disease, acts to suppress the establishment of both transgene-induced and virus-induced posttranscriptional gene silencing. The viral suppressor of silencing comprises the 5′-proximal region of the tobacco etch potyviral genomic RNA encoding P1, helper component-proteinase (HC-Pro) and a small part of P3, and is termed the P1/HC-Pro sequence. A reversal of silencing assay was used to assess the effect of the P1/HC-Pro sequence on transgenic tobacco plants (line T4) that are posttranscriptionally silenced for the uidA reporter gene. Silencing was lifted in offspring of T4 crosses with four independent transgenic lines expressing P1/HC-Pro, but not in offspring of control crosses. Viral vectors were used to assess the effect of P1/HC-Pro expression on virus-induced gene silencing (VIGS). The ability of a potato virus X vector expressing green fluorescent protein to induce silencing of a green fluorescent protein transgene was eliminated or greatly reduced when P1/HC-Pro was expressed from the same vector or from coinfecting potato virus X vectors. Expression of the HC-Pro coding sequence alone was sufficient to suppress virus-induced gene silencing, and the HC-Pro protein product was required for the suppression. This discovery points to the role of gene silencing as a natural antiviral defense system in plants and offers different approaches to elucidate the molecular basis of gene silencing.

A rapid Agrobacterium tumefaciens-mediated transformation system for wheat was developed using freshly isolated immature embryos, precultured immature embryos, and embryogenic calli as explants. The explants were inoculated with a disarmed A. tumefaciens strain C58 (ABI) harboring the binary vector pMON18365 containing the beta-glucuronidase gene with an intron, and a selectable marker, the neomycin phosphotransferase II gene. Various factors were found to influence the transfer-DNA delivery efficiency, such as explant tissue and surfactants present in the inoculation medium. The inoculated immature embryos or embryogenic calli were selected on G418-containing media. Transgenic plants were regenerated from all three types of explants. The total time required from inoculation to the establishment of plants in soil was 2.5 to 3 months. So far, more than 100 transgenic events have been produced. Almost all transformants were morphologically normal. Stable integration, expression, and inheritance of the transgenes were confirmed by molecular and genetic analysis. One to five copies of the transgene were integrated into the wheat genome without rearrangement. Approximately 35% of the transgenic plants received a single copy of the transgenes based on Southern analysis of 26 events. Transgenes in T1 progeny segregated in a Mendelian fashion in most of the transgenic plants

The carotenoid biosynthetic pathway in higher plants was manipulated by using an RNA viral vector. A cDNA encoding phytoene synthase and a partial cDNA encoding phytoene desaturase (PDS) were placed under the transcriptional control of a tobamovirus subgenomic promoter. One to two weeks after inoculation, systemically infected Nicotiana benthamiana plants were analyzed for phytoene. Leaves from transfected plants expressing phytoene synthase developed a bright orange phenotype and accumulated high levels of phytoene. Cytoplasmic inhibition of plant gene expression by viral RNA was demonstrated with an antisense RNA transcript to a partial PDS cDNA derived from tomato. The leaves of the plants transfected with the antisense PDS sequence developed a white phenotype and also accumulated high levels of phytoene. A partial cDNA to the corresponding N. benthamiana PDS gene was isolated and found to share significant homology with the tomato antisense PDS transcript. This work demonstrates that an episomal RNA viral vector can be used to deliberately manipulate a major, eukaryotic biosynthetic pathway. In addition, our results indicate that an antisense transcript generated in the cytoplasm of a plant cell can turn off endogenous gene expression.

As an important antioxidant for plants and humans, L-ascorbic acid (AsA) can scavenge reactive oxygen species and can be regenerated from its oxidized form in a reaction catalyzed by dehydroascorbate reductase (DHAR). To analyse the effect of overexpressing DHAR on tomato, an expression vector containing potato cytosolic DHAR (DHAR1) or chloroplastic DHAR (DHAR2) cDNA driven by a cauliflower mosaic virus 35S promoter was transferred into tomato plants. Compared with the wild type (WT), DHAR1 overexpression increased DHAR activity and AsA content in both leaves and fruits, while DHAR2 overexpression increased DHAR activity and AsA content mainly in leaves. DHAR1 and DHAR2 overexpression increased the chlorophyll content and photosynthetic rate of transgenic lines, but had no effect on plant height and stem diameter. Furthermore, the germination rate, plant fresh weight, seedling length and chlorophyll content of transgenic DHAR1 and DHAR2 plants under salt stress were higher than those of WT plants. In addition, the transgenic plants also exhibited considerable tolerance to oxidative damage induced by methyl viologen (MV). Taken together, these results indicated that overexpressing potato DHAR1 and DHAR2 enhanced the level of AsA in tomato and, consequently, increased the tolerance of tomato to salt and MV stress.

The ob/ob mouse is genetically deficient in leptin and exhibits a phenotype that includes obesity and non-insulin-dependent diabetes mellitus. This phenotype closely resembles the morbid obesity seen in humans. In this study, we demonstrate that a single intramuscular injection of a recombinant adeno-associated virus (AAV) vector encoding mouse leptin (rAAV-leptin) in ob/ob mice leads to prevention of obesity and diabetes. The treated animals show normalization of metabolic abnormalities including hyperglycemia, insulin resistance, impaired glucose tolerance, and lethargy. The effects of a single injection have lasted through the 6-month course of the study. At all time points measured the circulating levels of leptin in the serum were similar to age-matched control C57 mice. These results demonstrate that maintenance of normal levels of leptin (2-5 ng/ml) in the circulation can prevent both the onset of obesity and associated non-insulin-dependent diabetes. Thus a single injection of a rAAV vector expressing a therapeutic gene can lead to complete and long-term correction of a genetic disorder. Our study demonstrates the long-term correction of a disease caused by a genetic defect and proves the feasibility of using rAAV-based vectors for the treatment of chronic disorders like obesity

Great efforts are currently being devoted to studying the use of transgenes to confer resistance to phytopathogenic fungi. Snakin-1 is a broad-spectrum antimicrobial peptide isolated from Solanum that is active in vitro against bacteria and fungi. In this work wheat transgenic plants that constitutively expressed the S. chacoense SN1 gene were challenged with Blumeria graminis f.sp. tritici. Enhanced resistance to the pathogen was observed in two transgenic lines in which the development of the disease was delayed and reduced compared with the wild type variety ProINTA Federal. An association between high resistance to the pathogen and a high level of snakin-1 transcripts in the plant was observed. This is the first report on SN1 gene expression in Gramineae and its effects on wheat powdery mildew development.

Polygalacturonase-inhibiting proteins (PGIPs) selectively inhibit polygalacturonases (PGs) secreted by invading plant pathogenic fungi. The objective of the present research was to clone and introduce the pgip2 gene from Phaseolus vulgaris cv. Goli, with antifungal potential, into Brassica napus (R line Hyola 308) via Agrobacterium tumefaciens mediated transformation. Here we used a transgenic overexpression approach in order to investigate the inhibitory activity of the PGIP on the PG from Rhizoctonia solani. PGIP expression was determined in the functional inhibition assays against fungal PGs. Crude protein extracts prepared from transgenic canola leaves were found to inhibit the R. solani PG from 29% to 37% as compared to untransformed plants. The putative transgenic canola lines harbouring the pgip2 gene encoding polygalacturonase-inhibiting proteins were identified by polymerase chain reaction and Southern blot analysis.

:  Transgenic technology has been widely applied to a variety of freshwater fish species. However there are few reports on the use of this technology in commercially important marine species. In this study, the construction of expression vectors containing the β‐actin promoter region for use in the red sea bream Pagrus major, a species of considerable importance to the aquaculture industry in Japan is reported. The β‐actin gene was cloned from a red sea bream genomic DNA library. Recombinant plasmids were constructed by linking the 5′ flanking region of the β‐actin gene to the green fluorescent protein reporter gene, followed by the poly A signal sequence of simian virus 40 or the 3′ flanking region the β‐actin gene. Expression of these constructs was examined following microinjection into zebrafish and red sea bream embryos, and compared to that of the expression vector pXI‐GFP driven by the Xenopus elongation factor 1α. The results indicated that the construct consisting of the β‐actin 5′‐and 3′ flanking regions was the most efficacious. In future studies, it is planned to investigate the efficient condition for integration into chromosomes of the transgene.

The high-K(m) glucose transporter, GLUT-2, and the high-K(m) hexokinase of beta cells, glucokinase (GK), are required for glucose-stimulated insulin secretion (GSIS). GLUT-2 expression in beta cells of Zucker diabetic fatty (ZDF) rats is profoundly reduced at the onset of beta-cell dysfunction of diabetes. Because ZDF rats are homozygous for a mutation in their leptin receptor (OB-R) gene and are therefore leptin-insensitive, we expressed the wild-type OB-R gene in diabetic islets by infusing a recombinant adenovirus (AdCMV-OB-Rb) to determine whether this reversed the abnormalities. Leptin induced a rise in phosphorylated STAT3, indicating that the transferred wild-type OB-R was functional. GLUT-2 protein rose 17-fold in AdCMV-OB-Rb-treated ZDF islets without leptin, and leptin caused no further rise. GK protein rose 7-fold without and 12-fold with leptin. Preproinsulin mRNA increased 64% without leptin and rose no further with leptin, but leptin was required to restore GSIS. Clofibrate and 9-cis-retinoic acid, the partner ligands for binding to peroxisome proliferator-activator receptor alpha (PPAR alpha) and retinoid X receptor, up-regulated GLUT-2 expression in islets of normal rats, but not in ZDF rats, in which PPAR alpha is very low. Because the fat content of islets of diabetic ZDF rats remains high unless they are treated with leptin, it appears that restoration of GSIS requires normalization of intracellular nutrient homeostasis, whereas up-regulation of GLUT-2 and GK is leptin-independent, requiring only high expression of OB-Rb

TAIL-PCR is a powerful tool for the recovery of DNA fragments adjacent to known sequences. A protocol is presented for the amplification of insert end sequences from bacterial artificial chromosome clones using TAIL-PCR. The amplified products are suitable as probes for chromosome walking and genome mapping and as templates for direct sequencing. The protocol has been used in rice genome studies. Abbreviations: AD primer, arbitrary degenerate primer; BAC, bacterial artificial chromosome.