Impact of nitrogen and phosphorus fertilizers on Cry1Ac protein contents in transgenic cotton / Impacto de fertilizantes de nitrogênio e fósforo nos conteúdos de proteína Cry1Ac em algodão transgênico

Application of different fertilizers to check the efficiency of expression of Bt (Bacillus thuringiensis) gene in one of the leading commercialized crops (cotton) against Lepidopteran species is of great concern. The expression of Cry protein level can be controlled by the improvement of nutrients levels. Therefore, the myth of response of Cry toxin to different combinations of NP fertilizers was explored in three Bt cotton cultivars. Combinations include three levels of nitrogen and three levels of phosphorus fertilizers. Immunostrips and Cry gene(s) specific primer based PCR (Polymerase Chain Reaction) analysis were used for the presence of Bt gene that unveiled the presence of Cry1Ac gene only. Further, the ELISA (enzyme-linked immunosorbent assay) kit was used to quantify the expression of Cry1Ac protein. Under various NP fertilizers rates, the level of toxin protein exhibited highly significant differences. The highest toxin level mean was found to be 2.3740 and 2.1732 µg/g under the treatment of N150P75 kg ha-¹ combination while the lowest toxin level mean was found to be 0.9158 and 0.7641 µg/g at the N50P25 kg ha-¹ level at 80 and 120 DAS (Days After Sowing), respectively. It was concluded from the research that the usage of NP fertilizers has a positive relation with the expression of Cry1Ac toxin in Bt cotton. We recommend using the N150P50 kg ha-1 level as the most economical and practicable fertilizer instead of the standard dose N100P50 kg ha-¹ to get the desired level of Cry1Ac level for long lasting plant resistance (<1.5). The revised dose of fertilizer may help farmers to avoid the cross-resistance development in contradiction of insect pests.

A aplicação de diferentes fertilizantes para verificar a eficiência da expressão do gene Bt (Bacillus thuringiensis) em uma das principais culturas comercializadas (algodão) contra espécies de lepidópteros é uma grande preocupação. A expressão do nível de proteína Cry pode ser controlada pela melhoria dos níveis de nutrientes. Portanto, o mito da resposta da toxina Cry a diferentes combinações de fertilizantes NP foi explorado em três cultivares de algodão Bt. As combinações incluem três níveis de nitrogênio e três níveis de fertilizantes de fósforo. A análise de PCR (reação em cadeia da polimerase) específica para o gene (s) Immunostrips e Cry (s) foi usada para a presença do gene Bt que revelou a presença do gene Cry1Ac apenas. Além disso, o kit ELISA (ensaio de imunoabsorção enzimática) foi usado para quantificar a expressão da proteína Cry1Ac. Sob várias taxas de fertilizantes NP, o nível de proteína de toxina exibiu diferenças altamente significativas. A média do nível mais alto de toxina foi de 2,3740 e 2,1732 µg / g sob o tratamento da combinação N150P75 kg ha-¹, enquanto a média do nível mais baixo de toxina foi de 0,9158 e 0,7641 µg / g no nível de N50P25 kg ha-¹ em 80 e 120 DAS (dias após a semeadura), respectivamente. Concluiu-se com a pesquisa que o uso de fertilizantes NP tem relação positiva com a expressão da toxina Cry1Ac no algodão Bt. Recomendamos o uso do nível de N150P50 kg ha-¹ como o fertilizante mais econômico e praticável em vez da dose padrão N100P50 kg ha-¹ para obter o nível desejado de nível de Cry1Ac para resistência de planta de longa duração (<1,5). A dose revisada de fertilizante pode ajudar os agricultores a evitar o desenvolvimento de resistência cruzada em contradição com as pragas de insetos.

Impact of nitrogen and phosphorus fertilizers on Cry1Ac protein contents in transgenic cotton

Abstract Application of different fertilizers to check the efficiency of expression of Bt (Bacillus thuringiensis) gene in one of the leading commercialized crops (cotton) against Lepidopteran species is of great concern. The expression of Cry protein level can be controlled by the improvement of nutrients levels. Therefore, the myth of response of Cry toxin to different combinations of NP fertilizers was explored in three Bt cotton cultivars. Combinations include three levels of nitrogen and three levels of phosphorus fertilizers. Immunostrips and Cry gene(s) specific primer based PCR (Polymerase Chain Reaction) analysis were used for the presence of Bt gene that unveiled the presence of Cry1Ac gene only. Further, the ELISA (enzyme-linked immunosorbent assay) kit was used to quantify the expression of Cry1Ac protein. Under various NP fertilizers rates, the level of toxin protein exhibited highly significant differences. The highest toxin level mean was found to be 2.3740 and 2.1732 µg/g under the treatment of N150P75 kg ha-1 combination while the lowest toxin level mean was found to be 0.9158 and 0.7641 µg/g at the N50P25 kg ha-1 level at 80 and 120 DAS (Days After Sowing), respectively. It was concluded from the research that the usage of NP fertilizers has a positive relation with the expression of Cry1Ac toxin in Bt cotton. We recommend using the N150P50 kg ha-1 level as the most economical and practicable fertilizer instead of the standard dose N100P50 kg ha-1 to get the desired level of Cry1Ac level for long lasting plant resistance ( 1.5). The revised dose of fertilizer may help farmers to avoid the cross-resistance development in contradiction of insect pests.

Resumo A aplicação de diferentes fertilizantes para verificar a eficiência da expressão do gene Bt (Bacillus thuringiensis) em uma das principais culturas comercializadas (algodão) contra espécies de lepidópteros é uma grande preocupação. A expressão do nível de proteína Cry pode ser controlada pela melhoria dos níveis de nutrientes. Portanto, o mito da resposta da toxina Cry a diferentes combinações de fertilizantes NP foi explorado em três cultivares de algodão Bt. As combinações incluem três níveis de nitrogênio e três níveis de fertilizantes de fósforo. A análise de PCR (reação em cadeia da polimerase) específica para o gene (s) Immunostrips e Cry (s) foi usada para a presença do gene Bt que revelou a presença do gene Cry1Ac apenas. Além disso, o kit ELISA (ensaio de imunoabsorção enzimática) foi usado para quantificar a expressão da proteína Cry1Ac. Sob várias taxas de fertilizantes NP, o nível de proteína de toxina exibiu diferenças altamente significativas. A média do nível mais alto de toxina foi de 2,3740 e 2,1732 µg / g sob o tratamento da combinação N150P75 kg ha-1, enquanto a média do nível mais baixo de toxina foi de 0,9158 e 0,7641 µg / g no nível de N50P25 kg ha-1 em 80 e 120 DAS (dias após a semeadura), respectivamente. Concluiu-se com a pesquisa que o uso de fertilizantes NP tem relação positiva com a expressão da toxina Cry1Ac no algodão Bt. Recomendamos o uso do nível de N150P50 kg ha-1 como o fertilizante mais econômico e praticável em vez da dose padrão N100P50 kg ha-1 para obter o nível desejado de nível de Cry1Ac para resistência de planta de longa duração ( 1,5). A dose revisada de fertilizante pode ajudar os agricultores a evitar o desenvolvimento de resistência cruzada em contradição com as pragas de insetos.

Impact of nitrogen and phosphorus fertilizers on Cry1Ac protein contents in transgenic cotton / Impacto de fertilizantes de nitrogênio e fósforo nos conteúdos de proteína Cry1Ac em algodão transgênico

Abstract Application of different fertilizers to check the efficiency of expression of Bt (Bacillus thuringiensis) gene in one of the leading commercialized crops (cotton) against Lepidopteran species is of great concern. The expression of Cry protein level can be controlled by the improvement of nutrients levels. Therefore, the myth of response of Cry toxin to different combinations of NP fertilizers was explored in three Bt cotton cultivars. Combinations include three levels of nitrogen and three levels of phosphorus fertilizers. Immunostrips and Cry gene(s) specific primer based PCR (Polymerase Chain Reaction) analysis were used for the presence of Bt gene that unveiled the presence of Cry1Ac gene only. Further, the ELISA (enzyme-linked immunosorbent assay) kit was used to quantify the expression of Cry1Ac protein. Under various NP fertilizers rates, the level of toxin protein exhibited highly significant differences. The highest toxin level mean was found to be 2.3740 and 2.1732 µg/g under the treatment of N150P75 kg ha-1 combination while the lowest toxin level mean was found to be 0.9158 and 0.7641 µg/g at the N50P25 kg ha-1 level at 80 and 120 DAS (Days After Sowing), respectively. It was concluded from the research that the usage of NP fertilizers has a positive relation with the expression of Cry1Ac toxin in Bt cotton. We recommend using the N150P50 kg ha-1 level as the most economical and practicable fertilizer instead of the standard dose N100P50 kg ha-1 to get the desired level of Cry1Ac level for long lasting plant resistance (<1.5). The revised dose of fertilizer may help farmers to avoid the cross-resistance development in contradiction of insect pests.

Resumo A aplicação de diferentes fertilizantes para verificar a eficiência da expressão do gene Bt (Bacillus thuringiensis) em uma das principais culturas comercializadas (algodão) contra espécies de lepidópteros é uma grande preocupação. A expressão do nível de proteína Cry pode ser controlada pela melhoria dos níveis de nutrientes. Portanto, o mito da resposta da toxina Cry a diferentes combinações de fertilizantes NP foi explorado em três cultivares de algodão Bt. As combinações incluem três níveis de nitrogênio e três níveis de fertilizantes de fósforo. A análise de PCR (reação em cadeia da polimerase) específica para o gene (s) Immunostrips e Cry (s) foi usada para a presença do gene Bt que revelou a presença do gene Cry1Ac apenas. Além disso, o kit ELISA (ensaio de imunoabsorção enzimática) foi usado para quantificar a expressão da proteína Cry1Ac. Sob várias taxas de fertilizantes NP, o nível de proteína de toxina exibiu diferenças altamente significativas. A média do nível mais alto de toxina foi de 2,3740 e 2,1732 µg / g sob o tratamento da combinação N150P75 kg ha-1, enquanto a média do nível mais baixo de toxina foi de 0,9158 e 0,7641 µg / g no nível de N50P25 kg ha-1 em 80 e 120 DAS (dias após a semeadura), respectivamente. Concluiu-se com a pesquisa que o uso de fertilizantes NP tem relação positiva com a expressão da toxina Cry1Ac no algodão Bt. Recomendamos o uso do nível de N150P50 kg ha-1 como o fertilizante mais econômico e praticável em vez da dose padrão N100P50 kg ha-1 para obter o nível desejado de nível de Cry1Ac para resistência de planta de longa duração (<1,5). A dose revisada de fertilizante pode ajudar os agricultores a evitar o desenvolvimento de resistência cruzada em contradição com as pragas de insetos.

Effects of transgenic Bacillus Thuringiensis maize (2A-7) on the growth and development in rats.

This study aimed to determine the effects of genetically modified insect-resistant maize (2A-7) on the growth and development in developing rats. Rats were fed a diet formulated with 2A-7 maize and were compared with rats fed a diet formulated with non-transgenic maize (CK group) and rats fed AIN-93G diet (BC group). 2A-7 maize was formulated into diets at ratios of 82.4% (H group) and 20.6% (L group); non-transgenic maize was formulated into diets at a ratio of 82.4%. From the first day of pregnancy, adult rats were divided into four groups and fed with the above four diets, respectively. Weaning on postnatal day 21, the diets of offspring were consistent with their parents. The results showed that body weight, hematology, serum biochemistry, organ weight, organ coefficients and allergenicity of offspring fed with 2A-7 maize were comparable with those in the CK and BC groups. In physiological and behavioral development experiments, there was no statistically significant difference among groups. Although mCry1Ab proteins were detected in organs and serum, no histopathological changes were observed among groups. In conclusion, A-7 maize cause no treatment-related adverse effects on offspring, indicating that 2A-7 maize is safe for developing rats.

[Effect of soil salinity on insecticidal protein expression in flower buds of Bt cotton.] / 盐胁迫对Bt棉棉蕾杀虫蛋白表达的影响.

To clarify the effects of soil salinity on the insect-resistance of flower buds in transgenic Bt cotton of Xinmian 33B (salt-sensitive) and Zhong 07 (salt-tolerant), five levels (0, 0.15%, 0.30%, 0.45% and 0.60%) of soil salinity were set to investigate the impacts of soil salinity on Bt protein content, relative expression of Bt gene, activities of nitrogen metabolism-related enzymes and substances in flower buds during flowering stage. The results showed that Bt protein contents of flower buds decreased with increasing soil salinity. The Bt protein contents of flower buds decreased significantly when the soil salinity level was above 0.30% in both cotton cutivars. Greater reduction of Bt protein content occurred under severe than moderate soil salinity stress. However, the relative expression of Cry1Ac gene increased in flower buds of both cultivars when the stress level increased. Greater decreases of Bt protein content of flower buds was observed in Xinmian 33B compared to Zhong 07 under the same salinity level. The cultivars with greater reduction in Bt protein contents of flower buds also had greater reduction in the soluble protein content, glutamate pyruvate transaminase (GPT) and glutamic oxaloacetic transaminase (GOT) activities, and greater increments in free amino acid contents, protease activities and peptidase activities. Therefore, the decrease of Bt protein content was caused by decreased synthesis and increased decomposition of protein in flower buds of Bt cotton under soil salinity stress.

Performance of Bt-susceptible and -heterozygous genotypes of Spodoptera frugiperda (J.E. Smith) possessing single- or dual-gene resistance alleles in sequential feedings of non-Bt and Cry1A.105/Cry2Ab2 maize leaf tissues.

We simulated larval feeding behavior in seed blends of non-Bt and Bt maize to determine if seed blends create more favorable conditions for heterozygous-resistant insects over their Bt-susceptible counterparts. Survival, growth, development, and progeny production of four genotypes of the fall armyworm, Spodoptera frugiperda, Bt-susceptible (aabb), Cry1A.105 heterozygous resistant (Aabb), Cry2Ab2 heterozygous resistant (aaBb), and Cry1A.105/Cry2Ab2 heterozygous resistant (AaBb), were evaluated in eight feeding sequences (Seq 1-8) of non-Bt and MON89034 Bt maize leaf tissue expressing the Cry1A.105 and Cry2Ab2 proteins. We report variation in the performance of the four genotypes across the feeding sequences and biological parameters measured. Three heterozygous genotypes generally outperformed the susceptible genotype in larval survival, pupation rate, pupal weight, and progeny production. The performance was greater for Aabb over aaBb, AaBb over Aabb or aaBb, in two of the feeding sequences. The findings of this study could have important implications in assessing the risk of seed blends as refuge plantings for Bt crop resistance management where resistance in the target pest is not functionally recessive.

Generation of insect-resistant and glyphosate-tolerant rice by introduction of a T-DNA containing two Bt insecticidal genes and an EPSPS gene.

Insect resistance and glyphosate tolerance have been two of the most important traits in the genetic improvement of various crops. In this study, two Bacillus thuringiensis (Bt) insecticidal genes, Cry1Ac and Cry1Ig, and a modified glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene (G10) were combined into a single transferred DNA (T-DNA) fragment and introduced into rice by Agrobacterium-mediated transformation. A transgenic line with single-copy T-DNA insertion named GAI-14 was found to be highly resistant to striped stem borer and rice leaf roller, and tolerant to glyphosate. Analysis of T-DNA border sequence suggested that the transgenes were inserted at the chromosome 3 and appeared to have not interrupted any known or putative genes. A field trial observed no significant difference in the basic agronomic traits between GAI-14 and the recipient rice.

QTL analysis on rice grain appearance quality, as exemplifying the typical events of transgenic or backcrossing breeding.

Rice grain shape and yield are usually controlled by multiple quantitative trait loci (QTL). This study used a set of F9-10 recombinant inbred lines (RILs) derived from a cross of Huahui 3 (Bt/Xa21) and Zhongguoxiangdao, and detected 27 QTLs on ten rice chromosomes. Among them, twelve QTLs responsive for grain shape/ or yield were mostly reproducibly detected and had not yet been reported before. Interestingly, the two known genes involved in the materials, with one insect-resistant Bt gene, and the other disease-resistant Xa21 gene, were found to closely link the QTLs responsive for grain shape and weight. The Bt fragment insertion was firstly mapped on the chromosome 10 in Huahui 3 and may disrupt grain-related QTLs resulting in weaker yield performance in transgenic plants. The introgression of Xa21 gene by backcrossing from donor material into receptor Minghui 63 may also contain a donor linkage drag which included minor-effect QTL alleles positively affecting grain shape and yield. The QTL analysis on rice grain appearance quality exemplified the typical events of transgenic or backcrossing breeding. The QTL findings in this study will in the future facilitate the gene isolation and breeding application for improvement of rice grain shape and yield.

Establishment of an Efficient Chloroplast Gene Transformation System in Sugar Beet(Beta vulgaris L.)and Obtainment of Insect and Herbicide Resistant Sugar Beet Plants / 生物化学与生物物理进展

Insects pests and weeds are the main factors that reduce the yield of sugar beet. Genetic engineering breeding is an effective method to breed insect-resisitant and herbicide-resisitant sugar beet. A transformation system for foreign genes in sugar beet chloroplast was established. The expression of the foreign genes can confers resistance in transgenic sugar beet plants to insects pests and weeds. The chloroplast transformation vector pSKARBt/bar, which carries Bt cry1Ac gene and bar gene expression cassettes, was constructed by using molecular method. The Bt gene expression cassette contained the 3.5 kb Bt cry1Ac gene under the control of psbA promoter and terminator cloned from sugar beet chloroplast genome. The bar gene expression cassette contained the bar gene, 16 S promoter and terminator cloned from sugar beet chloroplast genome, The atpB and rbcL gene cloned from sugar beet chloroplast genome were used as homologous fragment, the bar gene was the selective marker. Plasmid pSKARBt/bar were transformed into the petioles of sugar beet with particle bombardment method. The petioles were planced onto the shoot-inducing selection medium which contained spectinomycin (20 mg/L), 6-BA (1.5 mg/L) and NAA (0.2 mg/L) at first. And when the green shoots regenerated, the green shoots were transfered into the shoot-propagation medium for optimal shoot development which contained spectinomycin (20 mg/L) and 6-BA (0.5 mg/L) and NAA (1.0 mg/L) one subculture at 20-day intervals, and then the shoots were transfered into the shoot-propagation medium for optimal shoot development with herbicide (PPT 10 mg/L) several subcultures. The shoots were transfered into the root-induction medium with herbicide (PPT 10 mg/L) and the transgenic plants were obtained at last. The transgenic sugar beet plants were testsed by PCR and Southern blot. The results showed that the Bt gene and bar gene had been transferred into the chloroplast genome of sugar beet. The transgenic plants had tolerance to both PPT and bioassays testsed. The insecticidal activity (the mortality of larvaes was 33%～80%) and herbicide resistance of the transgenic plants indicated that the relevant protein had been expressed already in sugar beet. The study showed that the bar gene can also be used as an efficient selective marker gene besides antibiotic resistant markers in plant transformation. Efficient transformation system in sugar beet chloroplast had been established.

The Environmental Effects of Genetically Modified Crops Resistant to Insects: [review] / Efeitos Ambientais de Culturas Geneticamente Modificadas Resistentes a Insetos: revisão]

Cultivares transgênicas de várias culturas estão sendo utilizadas em escala comercial em muitos países. A área dedicada ao cultivo com plantas transgênicas resistentes às pragas em todo o mundo alcançou 13 milhões de hectares em 2001. As cultivares transgênicas proporcionam benefícios, mas também apresentam riscos potenciais. As avaliações do seu impacto no ambiente são conduzidas antes da sua aprovação para uso comercial, como requerido pelas normas de biossegurança. Nesta revisão, serão discutidas as conseqüências ecológicas potenciais do uso comercial na agricultura de cultivares geneticamente modificadas que apresentam resistência aos insetos-pragas. Também serão discutidos os impactos ambientais causados pelas mudanças nas práticas agrícolas, identificando-se falhas e oportunidades de pesquisa, considerando-se essa nova ferramenta tecnológica. Os comentários e análises serão baseados no conhecimento atual que se tem dos riscos e beneficios do uso de cultivares resistentes a insetos, geneticamente modificadas, dentro do contexto dos programas de manejo integrado de pragas tradicionais.

Transgenic crops are currently being cultivated on a commercial scale in many countries. The area devoted to transgenic pest resistant varieties worldwide reached 13 million hectares in 2001. These varieties offer valuable benefits but also pose potential risks. Assessments of their impact on the environment are conducted before they are approved for commercial use, as required by the regulatory biosafety frameworks. In this review, we discuss the potential ecological consequences of the commercial use in agriculture of genetically modified insect resistant crops. We also discuss the impacts caused by the change in agricultural practices, and attempt to identify gaps and possible opportunities for research, considering this new technological tool. We based our analysis and comments on the current knowledge of the risks and benefits of these genetically modified insect resistant crops, within the context of traditional insect management strategies.