Expression of a chimeric CaMV 35S Bacillus thuringiensis insecticidal protein gene in transgenic tobacco

Insecticidal transgenic tobacco plants containing a truncated Bacillus thuringiensis cryIA(b) crystal protein (ICP) gene expressed from the CaMV 35S promoter were analyzed for ICP gene expression under field and greenhouse conditions over the course of a growing season. We present new information on temporal and tissue-specific expression of a CaMV 35S/cryIA(b) gene. Levels of cryIA(b) protein and mRNA were compared in both homozygous and hemizygous lines throughout plant development. Levels of ICP mRNA and protein increased during plant development with a pronounced rise in expression at the time of flowering. Homozygous ICP lines produced higher levels of ICP than the corresponding hemizygous lines. ELISA analysis of different tissues in the tobacco plant showed ICP gene expression in most tissues with a predominance of ICP in older tissue. All transgenic ICP tobacco lines which were studied in the field and greenhouse contained 400 ng to 1 g ICP per gram fresh weight in leaves from the mid-section of the plant at flowering. The amounts of ICP produced by field lines were directly comparable to levels observed in greenhouse-grown plants.     

Characterization of cymbidium mosaic virus coat protein gene and its expression in transgenic tobacco plants

Cymbidium mosaic virus (CyMV) is the most prevalent virus infecting orchids. Here, we report the isolation of partial cDNA clones encoding the genomic RNA of CyMV. Like most of the polyadenylated monopartite positive-strand RNA viruses, the open reading frame (ORF) coding for the viral coat protein (CP) is located at the 3 end. The ORF predicts a polypeptide chain of 220 amino acids with a molecular weight of 23 600. Sequence comparison of this ORF to the CP sequences of potato virus X(PVX) and white clover mosaic virus (WCIMV) revealed a strong amino acid homology in the mid-portion of the CP, but the overall homology was low. The CyMV CP gene was placed downstream of a cauliflower mosaic virus 35S promoter and the chimaeric gene was transferred into Nicotiana benthamiana. Transgenic plants expressing the CyMV CP were protected against CyMV infection.     

Agrobacterium and plant genetic engineering

Erratum

Agrobacterium and plant genetic engineering     

Tumorigenesis in transgenic mice: identification and characterization of synergizing oncogenes.

Transgenic mice carrying oncogenes present a useful model with which to assess the tissue-specific action of oncogenes. These mice are usually predisposed to a specific type of neoplastic growth. The tumors that arise are usually monoclonal in origin and become only apparent after a variable latency period, suggesting that additional events are required for tumor formation. Identification of these additional events is highly relevant: it might give access to the genes that can synergize with a preselected oncogene in tumorigenesis and could facilitate the identification of the biochemical pathways in which these genes act. Retroviruses can be instrumental in identifying cooperating oncogenes. Proto-oncogene activation or tumor suppressor gene inactivation by insertional mutagenesis is an important mechanism by which the non-acute transforming retroviruses can induce tumors in several species. Owing to the sequence tag provided by the provirus, the relevant proto-oncogene can be directly identified by cloning of the DNA flanking the proviral insertion site. We have exploited this potential of retroviruses by infecting E mu-pim-1 and E mu-myc transgenic mice, which are predisposed to lymphomagenesis, with Moloney murine leukemia virus (MuLV). A strong acceleration of tumor induction ensued upon infection of these mice with MuLV. More importantly, it allowed us to identify a number of additional common insertion sites marking both previously known as well as new (putative) oncogenes. In a significant portion of the tumors more than one oncogene was found to be activated, indicating that within this system the synergistic effect of at least three genes can be established.(ABSTRACT TRUNCATED AT 250 WORDS)     

Blastocystis hominis: pathogenic potential in human patients and in gnotobiotes.

Germfree guinea pigs were inoculated orally, in some experiments, and intracecally, in others, with Blastocystis hominis and the enteric flora from symptomatic patients. Other germfree guinea pigs received the parasite from axenic culture and still others from monoxenic culture with Proteus vulgaris. Fourteen of 43 animals inoculated orally with B. hominis and patient's enteric flora developed B. hominis infections and those with particularly heavy infections developed watery diarrhea of more than 1 week's duration immediately prior to sacrifice. Similar results were obtained from intracecal inoculations in that 13 of 28 animals developed infections and those with the greatest numbers of B. hominis had watery diarrhea for more than 1 week prior to sacrifice. Gross pathologic changes in these animals were mostly unremarkable, with only a slight hyperemia observed in several of the symptomatic animals. Microscopic examination, however, revealed frequent penetration of intestinal epithelium by B. hominis and the parasites in significant numbers were observed within the epithelium. There was a slight increase in cellularity in the lamina propria but parasites were not observed therein and their presence in the epithelium did not provoke an inflammatory response. Only one of eight animals inoculated from monoxenic cultures developed B. hominis infection (asymptomatic), and infections were not produced in animals inoculated from axenic culture. As a result of our observations of diarrhea in patients with particularly heavy infections with B. hominis together with the demonstration of similar symptoms in animals heavily infected with this parasite, we believe B. hominis may occasionally be related causally to the production of such symptoms by a mechanism not completely understandable.     

Comparison of bacterial and plant genes participating in proline biosynthesis with Osmotin gene, with respect to enhancing salinity tolerance of transgenic tobacco plants

In an attempt to increase tolerance to salinity stress in tobacco plants, the genes encoding the mutant form of glutamyl kinase (proB), pyrroline-5-carboxylate synthetase, and osmotin were cloned into three different shuttle vectors and were separately introduced into the tobacco plants. The transgenic lines were compared for their ability to produce shoots and grow in MS medium containing 320 mM NaCl; it was shown that the transgenic lines containing genetically handled osmotin gene are more resistant to salinity. The amount of chlorophyll a was used to show continuing growth of plant lines. The results showed that only the tobacco lines transformed with the modified osmotin gene exhibited greater tolerance to salinity. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 1, pp. 122–127. The text was submitted by the authors in English.     

Increased Rubisco content in maize mitigates chilling stress and speeds recovery

Many C₄ plants, including maize, perform poorly under chilling conditions. This phenomenon has been linked in part to decreased Rubisco abundance at lower temperatures. An exception to this is chilling‐tolerant Miscanthus, which is able to maintain Rubisco protein content under such conditions. The goal of this study was to investigate whether increasing Rubisco content in maize could improve performance during or following chilling stress. Here, we demonstrate that transgenic lines overexpressing Rubisco large and small subunits and the Rubisco assembly factor RAF1 (RAF1‐LSSS), which have increased Rubisco content and growth under control conditions, maintain increased Rubisco content and growth during chilling stress. RAF1‐LSSS plants exhibited 12% higher CO₂ assimilation relative to nontransgenic controls under control growth conditions, and a 17% differential after 2 weeks of chilling stress, although assimilation rates of all genotypes were ~50% lower in chilling conditions. Chlorophyll fluorescence measurements showed RAF1‐LSSS and WT plants had similar rates of photochemical quenching during chilling, suggesting Rubisco may not be the primary limiting factor that leads to poor performance in maize under chilling conditions. In contrast, RAF1‐LSSS had improved photochemical quenching before and after chilling stress, suggesting that increased Rubisco may help plants recover faster from chilling conditions. Relatively increased leaf area, dry weight and plant height observed before chilling in RAF1‐LSSS were also maintained during chilling. Together, these results demonstrate that an increase in Rubisco content allows maize plants to better cope with chilling stress and also improves their subsequent recovery, yet additional modifications are required to engineer chilling tolerance in maize.