A Built-In Strategy for Containment of Transgenic Plants: Creation of Selectively Terminable Transgenic Rice

Plant transgenic technology has been widely utilized for engineering crops for trait improvements and for production of high value proteins such as pharmaceuticals. However, the unintended spreading of commercial transgenic crops by pollination and seed dispersal is a major concern for environmental and food safety. Simple and reliable containment strategies for transgenes are highly desirable. Here we report a novel method for creating selectively terminable transgenic rice. In this method, the gene(s) of interest is tagged with a RNA interference cassette, which specifically suppresses the expression of the bentazon detoxification enzyme CYP81A6 and thus renders transgenic rice to be sensitive to bentazon, a herbicide used for rice weed control. We generated transgenic rice plants by this method using a new glyphosate resistant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from Pesudomonas putida as the gene of interest, and demonstrated that these transgenic rice plants were highly sensitive to bentazon but tolerant to glyphosate, which is exactly the opposite of conventional rice. Field trial of these transgenic rice plants further confirmed that they can be selectively killed at 100% by one spray of bentazon at a regular dose used for conventional rice weed control. Furthermore, we found that the terminable transgenic rice created in this study shows no difference in growth, development and yield compared to its non-transgenic control. Therefore, this method of creating transgenic rice constitutes a novel strategy of transgene containment, which appears simple, reliable and inexpensive for implementation

Molecular Dynamics Analysis Reveals Structural Insights into Mechanism of Nicotine N-Demethylation Catalyzed by Tobacco Cytochrome P450 Mono-Oxygenase

CYP82E4, a cytochrome P450 monooxygenase, has nicotine N-demethylase (NND) activity, which mediates the bioconversion of nicotine into nornicotine in senescing tobacco leaves. Nornicotine is a precursor of the carcinogen, tobacco-specific nitrosamine. CYP82E3 is an ortholog of CYP82E4 with 95% sequence identity, but it lacks NND activity. A recent site-directed mutagenesis study revealed that a single amino acid substitution, i.e., cysteine to tryptophan at the 330 position in the middle of protein, restores the NND activity of CYP82E3 entirely. However, the same amino acid change caused the loss of the NND activity of CYP82E4. To determine the mechanism of the functional turnover of the two molecules, four 3D structures, i.e., the two molecules and their corresponding cys–trp mutants were modeled. The resulting structures exhibited that the mutation site is far from the active site, which suggests that no direct interaction occurs between the two sites. Simulation studies in different biological scenarios revealed that the mutation introduces a conformation drift with the largest change at the F-G loop. The dynamics trajectories analysis using principal component analysis and covariance analysis suggests that the single amino acid change causes the opening and closing of the transfer channels of the substrates, products, and water by altering the motion of the F-G and B-C loops. The motion of helix I is also correlated with the motion of both the F-G loop and the B-C loop and; the single amino acid mutation resulted in the curvature of helix I. These results suggest that the single amino acid mutation outside the active site region may have indirectly mediated the flexibility of the F-G and B-C loops through helix I, causing a functional turnover of the P450 monooxygenase

Conversion of nicotine to nornicotine in Nicotiana tabacum is mediated by CYP82E4, a cytochrome P450 monooxygenase

Nornicotine is a secondary tobacco alkaloid that is produced by the N-demethylation of nicotine. Nornicotine production and accumulation in tobacco are undesirable because nornicotine serves as the precursor in the synthesis of the well characterized carcinogen N′-nitrosonornicotine during the curing and processing of tobacco. Although nornicotine is typically a minor alkaloid in tobacco plants, in many tobacco populations a high percentage of individuals can be found that convert a substantial proportion of the nicotine to nornicotine during leaf senescence and curing. We used a microarray-based strategy to identify genes that are differentially regulated between closely related tobacco lines that accumulate either nicotine (nonconverters) or nornicotine (converters) as the predominant alkaloid in the cured leaf. These experiments led to the identification of a small number of closely related cytochrome P450 genes, designated the CYP82E2 family, whose collective transcript levels were consistently higher in converter versus nonconverter tobacco lines. RNA interference-induced silencing of the CYP82E2 gene family suppressed the synthesis of nornicotine in strong converter plants to levels similar to that observed in nonconverter individuals. Although each of the six identified members of the P450 family share >90% nucleotide sequence identity, sense expression of three selected isoforms revealed that only one (CYP82E4v1) was involved in the conversion of nicotine to nornicotine. Yeast expression analysis revealed that CYP82E4v1 functions as a nicotine demethylase. Identification of the gene(s) responsible for nicotine demethylation provides a potentially powerful tool toward efforts to minimize nornicotine levels, and thereby N′-nitrosonornicotine formation, in tobacco products

Similarities and differences between pediatric and adult patients with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease with highest prevalence among women of childbearing age. However, children younger than 16 years also can develop SLE (childhood-onset lupus/juvenile-type SLE). The aim of our study was to compare the clinical course of adult and pediatric-onset SLE. Data from 342 adult patients followed at the University of Debrecen, Hungary, and 79 children documented in the Hungarian National Pediatric SLE registry were analyzed using hospital medical records. Organ manifestations, laboratory parameters, and immunoserological characteristics were reviewed and the results were evaluated using SPSS for Windows software. Gender distribution was not significantly different between groups with disease starting in childhood vs adulthood. The prevalence of the following manifestations was significantly higher for pediatric than for adult-onset disease including: lupus nephritis (43% pediatric vs 26.4% for adult-onset), hematological disorders (57% vs 36.4%), photosensitivity (20% vs 9%), butterfly rash (61% vs 35.5%) and mucosal ulceration (11.4% vs 4%). For adult-onset SLE, neurological symptoms (30% vs 6%) and polyarthritis (86% vs 68%) occurred significantly more frequently than in children. Anti-SSA, anti-SSB and antiphospholipid antibodies were detected at significantly higher levels in adult-onset patients compared to those in pediatrics. Children were more commonly given high-dose intravenous immunoglobulin treatment (6.3% vs 0.6%) and mycophenolate mofetil (15.2% vs 5.3%) than adults. These results suggest that pediatric and adult-onset SLE differ in multiple aspects, and it is important to recognize these differences for optimal treatment and prognosis of these patients

Seletividade de herbicidas aplicados em pré-emergência da cultura da cana-de-açúcar (RB 867515) tratada com nematicidas Selectivity of herbicides applied on pre-emergence of nematicide-treated sugarcane crop (RB 867515)

A aplicação simultânea de duas ou mais classes de defensivos sobre uma cultura agrícola pode provocar toxicidade às plantas. Assim, conduziu-se um experimento com o objetivo de avaliar a seletividade de herbicidas à cultura da cana-de-açúcar (RB 867515), quando aplicados em condição de pré-emergência, em áreas previamente tratadas com nematicidas no sulco de plantio. O experimento foi desenvolvido em área comercial de produção de cana-de-açúcar, no município de Piracicaba - SP, entre abril de 2003 e julho de 2004. Os tratamentos resultaram da combinação entre nove fatores herbicidas e quatro fatores nematicidas. Os herbicidas usados no experimento foram: sulfentrazone, tebuthiuron, metribuzin, ametrina, diuron, clomazone, pendimethalin e diuron + hexazinone, além de uma testemunha capinada. Os nematicidas utilizados foram: carbofuran, terbufós, aldicarb e uma testemunha sem nematicida. Avaliou-se a fitotoxicidade das diferentes combinações aos 15, 30, 60 e 90 dias após a brotação, o rendimento (t ha-1) e os parâmetros tecnológicos qualitativos. Observou-se que a seletividade inicial dos herbicidas foi prejudicada em função da interação das diferentes classes de defensivos utilizadas. Os sintomas de fitotoxicidade foram agudos, e os casos mais evidentes se originaram da associação dos nematicidas com clomazone, pendimethalin e tebuthiuron. Contudo, os danos fitotóxicos não se refletiram em perdas significativas de rendimento ou de qualidade de colmos, fato esse que pode ter sido influenciado pela capacidade de recuperação de injúrias apresentada pela variedade RB 867515, pela ocorrência de altas infestações de nematóides ou pela elevada variabilidade experimental.<br>Simultaneous applications of two or more classes of herbicides on a crop may promote ,totoxicity in the plants. Therefore, an experiment was conducted to evaluate herbicide selectivity in sugarcane, when applied under pre-emergence condition, in areas previously treated with nematicides on the planting furrow. The experiment was carried out in a commercial sugarcane area in Piracicaba (SP), from April 2003 to July 2004. The treatments resulted from the combination of nine herbicide factors and four nematicide factors. The herbicides used in the experiment were: sulfentrazone, tebuthiuron, metribuzin, ametrina, diuron, clomazone, pendimethalin and diuron+hexazinone, besides a hand-weeded check. The nematicides used were: carbofuran, terbufos, aldicarb and a check without nematicide. Phytotoxicity of the different combinations at 15, 30, 60 and 90 days after sprouting, yield (t ha-1) and qualitative technological parameters were evaluated. It was observed that the initial selectivity of the herbicides was reduced as a function of the interaction between the different classes used. Phytotoxic symptoms were acute, with the most serious cases originating from the association of nematicides with clomazone, pendimethalin and tebuthiuron. However, phytotoxic damages did not reflect a significant yield or qualitative losses, a fact that may have influenced injury recovery capacity, as presented by the RB 867515 variety, due to high nematode infestations or high experimental variability