Proteomic Analysis of Bt cry1Ac Transgenic Oilseed Rape (Brassica napus L.).

Oilseed rape (Brassica napus L.) is an important cash crop, but transgenic oilseed rape has not been grown on a commercial scale in China. It is necessary to analyze the characteristics of transgenic oilseed rape before commercial cultivation. In our study, differential expression of total protein from the leaves in two transgenic lines of oilseed rape expressing foreign Bt Cry1Ac insecticidal toxin and their non-transgenic parent plant was analyzed using a proteomic approach. Only shared changes in both of the two transgenic lines were calculated. Fourteen differential protein spots were analyzed and identified, namely, eleven upregulated expressed protein spots and three downregulated protein spots. These proteins are involved in photosynthesis, transporter function, metabolism, protein synthesis, and cell growth and differentiation. The changes of these protein spots in transgenic oilseed rape may be attributable to the insertion of the foreign transgenes. However, the transgenic manipulation might not necessarily cause significant change in proteomes of the oilseed rape.

Foreign cry1Ac gene integration and endogenous borer stress-related genes synergistically improve insect resistance in sugarcane.

BACKGROUND: Sugarcane (Saccharum spp. hybrids) is considered the most globally important sugar-producing crop and raw material for biofuel. Insect attack is a major issue in sugarcane cultivation, resulting in yield losses and sucrose content reductions. Stem borer (Diatraea saccharalis F.) causes serious yield losses in sugarcane worldwide. However, insect-resistant germplasms for sugarcane are not available in any collections all over the world, and the molecular mechanism of insect resistance has not been elucidated. In this study, cry1Ac transgenic sugarcane lines were obtained and the biological characteristics and transgene dosage effect were investigated and a global exploration of gene expression by transcriptome analysis was performed. RESULTS: The transgene copies of foreign cry1Ac were variable and random. The correlation between the cry1Ac protein and cry1Ac gene copies differed between the transgenic lines from FN15 and ROC22. The medium copy lines from FN15 showed a significant linear relationship, while ROC22 showed no definite dosage effect. The transgenic lines with medium copies of cry1Ac showed an elite phenotype. Transcriptome analysis by RNA sequencing indicated that up/down regulated differentially expressed genes were abundant among the cry1Ac sugarcane lines and the receptor variety. Foreign cry1Ac gene and endogenous borer stress-related genes may have a synergistic effect. Three lines, namely, A1, A5, and A6, were selected for their excellent stem borer resistance and phenotypic traits and are expected to be used directly as cultivars or crossing parents for sugarcane borer resistance breeding. CONCLUSIONS: Cry1Ac gene integration dramatically improved sugarcane insect resistance. The elite transgenic offspring contained medium transgene copies. Foreign cry1Ac gene integration and endogenous borer stress-related genes may have a synergistic effect on sugarcane insect resistance improvement.

Subchronic toxicity study in rats evaluating genetically modified DAS-81419-2 soybean.

A 90-day feeding study in rats was conducted to evaluate the subchronic oral toxicity of genetically modified (GM) DAS-81419-2 soybean. Wistar rats were fed with diets containing toasted soybean meal produced from DAS-81419-2 soybean grain that expresses the Cry1F, Cry1Ac, and Pat proteins or containing conventional soybean at doses of 30.0%, 15.0%, 7.5%, or 0% (control group) for 90 consecutive days. The general behavior, body weight and food consumption were observed. At the middle and end of the experiment, blood, serum, and urine samples were collected for biochemical assays. At the conclusion of the study, the internal organs were weighed and histopathological examination was completed. The rats exhibited free movement and shiny coats without any abnormal symptoms or abnormal secretions in their noses, eyes, or mouths. There were no adverse effects on body weight in GM soybean groups and conventional soybean groups. No biological differences in hematological, biochemical, or urine indices were observed. No significant differences in relative organ weights were detected between the experimental groups and the control group. No histopathological changes were observed. Under the conditions of this study, DAS-81419-2 soybean did not cause any treatment-related effects in Wistar rats following 90 days of dietary administration.

Comparative proteomics of Bt-transgenic and non-transgenic cotton leaves.

BACKGROUND: As the rapid growth of the commercialized acreage in genetically modified (GM) crops, the unintended effects of GM crops' biosafety assessment have been given much attention. To investigate whether transgenic events cause unintended effects, comparative proteomics of cotton leaves between the commercial transgenic Bt + CpTI cotton SGK321 (BT) clone and its non-transgenic parental counterpart SY321 wild type (WT) was performed. RESULTS: Using enzyme linked immunosorbent assay (ELISA), Cry1Ac toxin protein was detected in the BT leaves, while its content was only 0.31 pg/g. By 2-DE, 58 differentially expressed proteins (DEPs) were detected. Among them 35 were identified by MS. These identified DEPs were mainly involved in carbohydrate transport and metabolism, chaperones related to post-translational modification and energy production. Pathway analysis revealed that most of the DEPs were implicated in carbon fixation and photosynthesis, glyoxylate and dicarboxylate metabolism, and oxidative pentose phosphate pathway. Thirteen identified proteins were involved in protein-protein interaction. The protein interactions were mainly involved in photosynthesis and energy metabolite pathway. CONCLUSIONS: Our study demonstrated that exogenous DNA in a host cotton genome can affect the plant growth and photosynthesis. Although some unintended variations of proteins were found between BT and WT cotton, no toxic proteins or allergens were detected. This study verified genetically modified operation did not sharply alter cotton leaf proteome, and the target proteins were hardly checked by traditional proteomic analysis.

Rhizospheric fungal community structure of a Bt brinjal and a near isogenic variety.

AIMS: The objective of this study was to investigate the influence of Cry1Ac gene expressing brinjal (VRBT-8) on the rhizospheric fungal community structure. METHODS AND RESULTS: qPCR indicated variations in the fungal ITS rRNA copy numbers of non-Bt (1·43-4·43) × 10(9) g(-1) dws and Bt (1·43-3·32) × 10(9) g(-1) dws plots. Phylogenetic analysis of ITS rRNA clones indicated fungal-related group majority of being Ascomycota compared to that of Basidiomycota and Zygomycota in non-Bt- and Bt-planted soils. Sordariomycetes was the dominant class detected in all the stages. CONCLUSIONS: Despite the variations in the population size and the distribution pattern observed across the non-Bt and Bt brinjal, plant-growth-dependent variability was more prominent compared with genetic modification. Therefore, this study concludes that genetic modification of brinjal crop has minor effect on the fungal community. SIGNIFICANCE AND IMPACT OF THE STUDY: Brinjal, the important solanaceous crop, is also prone to attack by many insect pests, especially by Leucinoides orbonalis, resulting in significant losses in the crop yield. However, the reports on the effect of transgenic crops and the associated microbial community are inconsistent. The present communication takes into account for the first time the possible interactions between Bt brinjal and the associated fungal community; the latter playing a significant role in maintaining soil fertility. As this study is limited to the structural diversity of fungal community, additional information regarding the functional diversity of the group seems imperative before recommending the commercialization of GM crops.

Transformation of coffee (Coffea Arabica L. cv. Catimor) with the cry1ac gene by biolistic, without the use of markers

The transformation of coffee plantlets with the cry1ac gene of Bacillus thuringiensis was achieved by biolistic using either the whole pUBC plasmid or only the ubi-cry1ac-nos genetic cassette. The cry1ac gene was inserted into coffee plants in order to confer resistance to the leaf miner Leucoptera coffeella, an insect responsible for considerable losses in coffee crops. Bearing in mind that the genetic cassettes used for this study lack reporter genes and/or selection marker genes, the parameters for the transformation procedure by biolistic were previously standardised with a plasmid carrying the gus reporter gene. The presence of the cry1ac gene in young plantlet tissues was determined by PCR, Southern blot and reverse transcription-PCR. Our results show that the obtainment of viable coffee plantlets, transformed by bombardment with the cry1ac gene and without selection markers nor reporter genes, is feasible.

A transformação das plântulas de café com o gene cry1ac de Bacillus thuringiensis foi realizada por biobalística, utilizando todo o pUBC plasmídeo ou só o cassete genético UBI-cry1ac-nos. O gene cry1ac foi inserido no cafeeiro a fim de conferir resistência à folha mineiro Leucoptera coffeella, um inseto responsável por perdas consideráveis nas culturas de café. Tendo em conta que ao plasmídeo e ao cassete genético utilizados para este estudo faltam genes repórteres e/ou de seleção, os parâmetros para o processo de transformação por biobalística foram previamente padronizados com um plasmídeo transportando o gene repórter gus. A presença do gene cry1ac em tecidos de jovens plântulas foi determinada por PCR, Southern blot e transcrição reversa-PCR. Nossos resultados mostram que a obtenção de plântulas de café, transformado por bombardeamento com o gene cry1ac sem genes de seleção genética nem repórteres é viável.

Transformation of coffee (Coffea Arabica L. cv. Catimor) with the cry1ac gene by biolistic, without the use of markers / Transformação do café (Coffea arabica L. cv. Catimor) com o gene cry1ac por bombardeamento, sem usar marcadores

The transformation of coffee plantlets with the cry1ac gene of Bacillus thuringiensis was achieved by biolistic using either the whole pUBC plasmid or only the ubi-cry1ac-nos genetic cassette. The cry1ac gene was inserted into coffee plants in order to confer resistance to the leaf miner Leucoptera coffeella, an insect responsible for considerable losses in coffee crops. Bearing in mind that the genetic cassettes used for this study lack reporter genes and/or selection marker genes, the parameters for the transformation procedure by biolistic were previously standardised with a plasmid carrying the gus reporter gene. The presence of the cry1ac gene in young plantlet tissues was determined by PCR, Southern blot and reverse transcription-PCR. Our results show that the obtainment of viable coffee plantlets, transformed by bombardment with the cry1ac gene and without selection markers nor reporter genes, is feasible.

A transformação das plântulas de café com o gene cry1ac de Bacillus thuringiensis foi realizada por biobalística, utilizando todo o pUBC plasmídeo ou só o cassete genético UBI-cry1ac-nos. O gene cry1ac foi inserido no cafeeiro a fim de conferir resistência à folha mineiro Leucoptera coffeella, um inseto responsável por perdas consideráveis nas culturas de café. Tendo em conta que ao plasmídeo e ao cassete genético utilizados para este estudo faltam genes repórteres e/ou de seleção, os parâmetros para o processo de transformação por biobalística foram previamente padronizados com um plasmídeo transportando o gene repórter gus. A presença do gene cry1ac em tecidos de jovens plântulas foi determinada por PCR, Southern blot e transcrição reversa-PCR. Nossos resultados mostram que a obtenção de plântulas de café, transformado por bombardeamento com o gene cry1ac sem genes de seleção genética nem repórteres é viável.

Resistance to Anticarsia gemmatalis Hübner (Lepidoptera, Noctuidae) in transgenic soybean (Glycine max (L) Merrill Fabales, Fabaceae) cultivar IAS5 expressing a modified Cry1Ac endotoxin

Somatic embryos of the commercial soybean (Glycine max) cultivar IAS5 were co-transformed using particle bombardment with a synthetic form of the Bacillus thuringiensis delta-endotoxin crystal protein gene cry1Ac, the beta-glucuronidase reporter gene gusA and the hygromycin resistance gene hpt. Hygromycin-resistant tissues were proliferated individually to give rise to nine sets of clones corresponding to independent transformation events. The co-bombardment resulted in a co-transformation efficiency of 44 percent. Many histodifferentiated embryos and 30 well-developed plants were obtained. Twenty of these plants flowered and fourteen set seeds. The integration and expression of the cry1Ac, gusA and hpt transgenes into the genomes of a sample of transformed embryos and all T0, T1, T2 and T3 plants were confirmed by Gus activity, PCR, Southern and western blot, and ELISA techniques. Two T0 plants out of the seven co-transformed plants produced seeds and were analyzed for patterns of integration and inheritance until the T3 generation. Bioassays indicated that the transgenic plants were highly toxic to the velvetbean caterpillar Anticarsia gemmatalis, thus offering a potential for effective insect resistance in soybean.

Genetically modified coffee plants expressing the Bacillus thuringiensis cry1Ac gene for resistance to leaf miner.

A synthetic version of the cry1Ac gene of Bacillus thuringiensis has been used for the transformation of coffee species (Coffea canephora and C. arabica) to confer resistance to an important pest, the coffee leaf miner (Perileucoptera coffeella and other Leucoptera spp). Somatic embryos were co-cultivated with the LBA4404 strain of Agrobacterium tumefaciens containing the cry1Ac gene. More than 100 transformed plants from independent transformation events were obtained for each coffee genotype. The integration and expression of the cry1Ac gene was studied, and effective resistance of transgenic plants against leaf miner was verified in bioassays with the insects. These plants could represent a good opportunity to analyse the impact of genetic engineering of perennial crops for sustainable resistance to an obligate endocarpic pest using a B. thuringiensis insecticidal protein.