Transgenic Populations Research Articles

Mouse Phenome Database: a data repository and analysis suite for curated primary mouse phenotype data.

The Mouse Phenome Database (MPD; https://phenome.jax.org) is a widely accessed and highly functional data repository housing primary phenotype data for the laboratory mouse accessible via APIs and providing tools to analyze and visualize those data. Data come from investigators around the world and represent a broad scope of phenotyping endpoints and disease-related traits in naïve mice and those exposed to drugs, environmental agents or other treatments. MPD houses rigorously curated per-animal data with detailed protocols. Public ontologies and controlled vocabularies are used for annotation. In addition to phenotype tools, genetic analysis tools enable users to integrate and interpret genome–phenome relations across the database. Strain types and populations include inbred, recombinant inbred, F1 hybrid, transgenic, targeted mutants, chromosome substitution, Collaborative Cross, Diversity Outbred and other mapping populations. Our new analysis tools allow users to apply selected data in an integrated fashion to address problems in trait associations, reproducibility, polygenic syndrome model selection and multi-trait modeling. As we refine these tools and approaches, we will continue to provide users a means to identify consistent, quality studies that have high translational relevance.

Increased Akt signaling in the fat body of Anopheles stephensi extends lifespan and increases lifetime fecundity through modulation of insulin-like peptides

Insulin-like peptides (ILPs) and the insulin/insulin-like growth factor 1 signaling (IIS) cascade regulate numerous physiological functions, including lifespan, reproduction, immunity, and metabolism, in diverse eukaryotes. We previously demonstrated that in female Anopheles stephensi and Aedes aegypti mosquitoes, activation of the IIS cascade in the fat body led to a significant increase in lifespan. In this work, we elucidated two putative mechanisms in A. stephensi behind the observed lifespan extension and assessed whether this lifespan extension confers an overall fitness advantage to the mosquito. Specifically, we demonstrated that increased Akt signaling in the mosquito fat body following a blood meal significantly suppressed the expression of ILP2 in the head. Moreover, overexpression of active Akt in the fat body altered the expression of a putative insulin binding protein ortholog, Imaginal morphogenesis protein-Late 2 (Imp-L2), in response to transgene expression. Combined, these two factors may act to reduce overall levels of circulating ILP2 or other ILPs in the mosquito, in turn conferring increased survival. We also examined the impact increased fat body IIS had on lifetime fecundity and demonstrated that transgenic female mosquito populations had higher lifetime fecundity relative to non-transgenic sibling controls. These studies provide new insights into the complex hormonal and molecular mechanisms regulating the interplay between IIS, aging, and reproduction in this important vector of human malaria parasites.

Interactive Effects of [CO2] and Temperature on Plant Chemistry of Transgenic Bt Rice and Population Dynamics of a Non-Target Planthopper, Nilaparvata lugens (Stål) under Different Levels of Soil Nitrogen.

Gaining a better understanding of the interactive effect of projected atmospheric CO2 level increase and the Earth’s rising temperature on plant chemistry (nutritional and defensive characteristics) of transgenic crops is essential when attempting to forecast the responses of target and non-target insects to climate change. In this study, effects of carbon dioxide (CO2; elevated versus ambient), temperature (T; high versus low), and their interactions on leaf nitrogen content (N%) and C:N ratio of transgenic Bt rice and its non-Bt isoline grown under low- and high-N fertilizer were systematically analyzed together with the resulting insect population dynamics of a non-target planthopper Nilaparvata lugens (Stâl) in open-top-chamber experiments. The results indicated that under low-N treatment, elevated CO2 at low T (i.e., eCO2) (compared to ambient CO2 at low T, i.e., CK) significantly decreased N% and Bt-toxin content and significantly increased C:N ratio in leaf sheath and leaf of Bt rice, especially during the tillering stage, whereas inverse effects of high T were shown on the plant chemistry of Bt rice, especially during heading stage. The combination of elevated CO2 and high T (i.e., Combined) (in contrast to CK) significantly increased N% and decreased C:N ratio in leaf sheath of Bt rice during the heading stage under low-N fertilizer, while significantly decreased N% and increased C:N ratio in leaf of Bt rice during the tillering stage, regardless of fertilizer-N level, and significantly increased Bt-toxin content in leaf sheath and leaf during the tillering stage under both low- and high-N. Moreover, no discernable relationships between Bt-toxin content and N% or leaf C:N ratio were observed at any CO2 or N levels evaluated. Furthermore, transgenic treatment, temperature and fertilizer-N level interactions, and CO2 and fertilizer-N level interactions all significantly affected the population dynamics of N. lugens. Specifically, high-N significantly enhanced the population dynamics of N. lugens fed on non-Bt rice grown under eTemp and Bt cultivar significantly reduced the population dynamics of N. lugens under eCO2 regardless of N fertilizer levels. The study demonstrates that the planting of transgenic Bt rice would not increase the risk of increased N. lugens severity under the combined condition of elevated CO2 and increased temperature, particularly under moderate level of N fertility.

A quick, easy and cost-effective in planta method to develop direct transformants in wheat.

Agrobacterium mediated in planta method was used to transform Indian elite wheat genotype HD2894 with herbicide-tolerant CP4-EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) gene. The apical meristems of germinated seeds were targeted for introgression of transgene. The obtained T1 plants were screened by spraying 1% glyphosate and only positive transformants survived. The presence of transgene was also confirmed by PCR and Southern hybridization. Using this method, 3.07% transformation rate was observed. To identify transgenic lines carrying stably integrated CP4-EPSPS gene, the transgenic populations were screened in T3 generation using 1% glyphosate and lines with 100% survival were considered as homozygous. No significant morpho-physiological variations were observed within the transgenic lines as compared to non-transgenic plants. The present study resulted in herbicide-tolerant transgenic wheat and provides a valuable tool for development of wheat genetic transformation.

Development of an Agrobacterium-delivered CRISPR/Cas9 system for wheat genome editing.

Summary CRISPR/Cas9 has been widely used for genome editing in many organisms, including important crops like wheat. Despite the tractability in designing CRISPR/Cas9, efficacy in the application of this powerful genome editing tool also depends on DNA delivery methods. In wheat, the biolistics based transformation is the most used method for delivery of the CRISPR/Cas9 complex. Due to the high frequency of gene silencing associated with co‐transferred plasmid backbone and low edit rate in wheat, a large T0 transgenic plant population are required for recovery of desired mutations, which poses a bottleneck for many genome editing projects. Here, we report an Agrobacterium‐delivered CRISPR/Cas9 system in wheat, which includes a wheat codon optimized Cas9 driven by a maize ubiquitin gene promoter and a guide RNA cassette driven by wheat U6 promoters in a single binary vector. Using this CRISPR/Cas9 system, we have developed 68 edit mutants for four grain‐regulatory genes, TaCKX2‐1, TaGLW7, TaGW2, and TaGW8, in T0, T1, and T2 generation plants at an average edit rate of 10% without detecting off‐target mutations in the most Cas9‐active plants. Homozygous mutations can be recovered from a large population in a single generation. Different from most plant species, deletions over 10 bp are the dominant mutation types in wheat. Plants homozygous of 1160‐bp deletion in TaCKX2‐D1 significantly increased grain number per spikelet. In conclusion, our Agrobacterium‐delivered CRISPR/Cas9 system provides an alternative option for wheat genome editing, which requires a small number of transformation events because CRISPR/Cas9 remains active for novel mutations through generations.

A Novel Dietary Source of EPA and DHA: Metabolic Engineering of an Important Freshwater Species—Common Carp by fat1-Transgenesis

Omega-3 polyunsaturated fatty acids (n-3 PUFAs), such as eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA), are essential for neural development and human health. The n-3 PUFAs are mainly obtained from marine fish by dietary intake. Freshwater fish species usually contain low level of n-3 PUFAs due to the lack of n-3 PUFAs in their food chain. In this study, we report on the substantial production of EPA and DHA in a globally important freshwater fish species, common carp (Cyprinus carpio). This was achieved by introducing an "all-fish" transgene CA:fat1 containing the fish codon-optimized omega-3 desaturase gene (fat1) driven by the common carp β-actin promoter (CA). Through a sperm sample screening method, we successfully generated fat1-positive F1 transgenic population with high efficiency. In F1 population, the muscle contents of ALA (18:3n-3), EPA and DHA were significantly increased when compared with non-transgenic siblings. Thereafter, four independent F2 heterozygous lines were obtained from 4 F1 transgenic males and a detailed comparison of fatty acids profile and growth performance was carried out for these 4 lines. All fat1-transgenic common carps from 4 lines showed an evident decrease in n-6 PUFA contents and a substantial increase in n-3 PUFA contents, among which line 4 stands out, showing a statistically significant increase in all 4 types of n-3 PUFAs including ALA (4.4-fold increase, p < 0.001), EPA (4.8-fold increase, p < 0.01), C22:5n-3 (DPA, 2.4-fold increase, p < 0.05), and DHA (1.9-fold increase, p < 0.05). Therefore, the line 4 was selected as the optimized breeding stock for further study, and the proximate nutrition composition and PUFA synthesis pathway were analyzed. Our study demonstrates that in the transgenic group, the muscular lipid content did not change, while fat accumulations in the internal organs and especially in the liver were significantly decreased as a result of hyperactivation of fatty acid oxidation process. Finally, we conclude that the "all-fish" CA:fat1-transgenic freshwater fish-common carp-can serve as a novel healthy dietary source of n3-PUFAs, especially EPA and DHA.

Modeling the spreading and interaction between wild and transgenic mosquitoes with a random dispersal.

Due to recent advances in genetic manipulation, transgenic mosquitoes may be a viable alternative to reduce some diseases. Feasibility conditions are obtained by simulating and analyzing mathematical models that describe the behavior of wild and transgenic populations living in the same geographic area. In this paper, we present a reaction–diffusion model in which the reaction term is a nonlinear function that describes the interaction between wild and transgenic mosquitoes, considering the zygosity, and the diffusive term that represents a nonuniform spatial spreading characterized by a random diffusion parameter. The resulting nonlinear system of partial differential equations is numerically solved using the sequential operator splitting technique, combining the finite element method and Runge-Kutta method. This scheme is numerically implemented considering uncertainty in the diffusion parameters of the model. Several scenarios simulating spatial release strategies of transgenic mosquitoes are analyzed, demonstrating an intrinsic association between the transgene frequency in the total population and the strategy adopted.

"Born to Run"? Not Necessarily: Species and Trait Bias in Persistent Free-Living Transgenic Plants.

The possibility of transgenes from engineered plants ending up in unmanaged populations with undesirable consequences has been a long-term biosafety concern. Experience with traditionally improved plants reveals that most cases of such gene escape have been of little consequence, but on occasion they have led to the evolution of problematic plants or have resulted in an increased extinction risk for wild taxa. Three decades have passed since the first environmental release of transgenic plants, and more than two decades since their first commercialization. Examples of transgenes gone astray are increasingly commonplace. Transgenic individuals have been identified in more than a thousand free-living plant populations. Here I review 14 well-documented consolidated “cases” in which transgenes have found their way into free-living plant populations. Some as transient volunteers; others appear to be persistent transgenic populations. The species involved in the latter are not representative of the current commercialized transgenic crops as whole. They tend to share certain traits that are absent or rare in the transgenic crops that do not exist as persistent populations. The traits commonly occurring in species with persistent transgenic free-living populations are the following, in descending order of importance: (1) a history of occurring as non-transgenic free-living plants, (2) fruits fully or partially shattering prior to harvest, (3) have small or otherwise easily dispersed seeds, either spontaneously or by seed spillage along the supply chain from harvest to consumer, (4) ability to disperse viable pollen, especially to a kilometer or more, (5) perennial habit, and (6) the transgene's fitness effects in the recipient environment are beneficial or neutral. Based on these observations, a thought experiment posits which species might be the next to be reported to occur as free-living transgenic populations.

Development and Evaluation of Low Phytic Acid Soybean by siRNA Triggered Seed Specific Silencing of Inositol Polyphosphate 6-/3-/5-Kinase Gene.

Soybean is one of the leading oilseed crop in the world and is showing a remarkable surge in its utilization in formulating animal feeds and supplements. Its dietary consumption, however, is incongruent with its existing industrial demand due to the presence of anti-nutritional factors in sufficiently large amounts. Phytic acid in particular raises concern as it causes a concomitant loss of indigestible complexed minerals and charged proteins in the waste and results in reduced mineral bioavailability in both livestock and humans. Reducing the seed phytate level thus seems indispensable to overcome the nutritional menace associated with soy grain consumption. In order to conceive our objective we designed and expressed a inositol polyphosphate 6-/3-/5-kinase gene-specific RNAi construct in the seeds of Pusa-16 soybean cultivar. We subsequently conducted a genotypic, phenotypic and biochemical analysis of the developed putative transgenic populations and found very low phytic acid levels, moderate accumulation of inorganic phosphate and elevated mineral content in some lines. These low phytic acid lines did not show any reduction in seedling emergence and displayed an overall good agronomic performance.

Modifier locus mapping of a transgenic F2 mouse population identifies CCDC115 as a novel aggressive prostate cancer modifier gene in humans

BackgroundIt is well known that development of prostate cancer (PC) can be attributed to somatic mutations of the genome, acquired within proto-oncogenes or tumor-suppressor genes. What is less well understood is how germline variation contributes to disease aggressiveness in PC patients. To map germline modifiers of aggressive neuroendocrine PC, we generated a genetically diverse F2 intercross population using the transgenic TRAMP mouse model and the wild-derived WSB/EiJ (WSB) strain. The relevance of germline modifiers of aggressive PC identified in these mice was extensively correlated in human PC datasets and functionally validated in cell lines.ResultsAggressive PC traits were quantified in a population of 30 week old (TRAMP x WSB) F2 mice (n = 307). Correlation of germline genotype with aggressive disease phenotype revealed seven modifier loci that were significantly associated with aggressive disease. RNA-seq were analyzed using cis-eQTL and trait correlation analyses to identify candidate genes within each of these loci. Analysis of 92 (TRAMP x WSB) F2 prostates revealed 25 candidate genes that harbored both a significant cis-eQTL and mRNA expression correlations with an aggressive PC trait. We further delineated these candidate genes based on their clinical relevance, by interrogating human PC GWAS and PC tumor gene expression datasets. We identified four genes (CCDC115, DNAJC10, RNF149, and STYXL1), which encompassed all of the following characteristics: 1) one or more germline variants associated with aggressive PC traits; 2) differential mRNA levels associated with aggressive PC traits; and 3) differential mRNA expression between normal and tumor tissue. Functional validation studies of these four genes using the human LNCaP prostate adenocarcinoma cell line revealed ectopic overexpression of CCDC115 can significantly impede cell growth in vitro and tumor growth in vivo. Furthermore, CCDC115 human prostate tumor expression was associated with better survival outcomes.ConclusionWe have demonstrated how modifier locus mapping in mouse models of PC, coupled with in silico analyses of human PC datasets, can reveal novel germline modifier genes of aggressive PC. We have also characterized CCDC115 as being associated with less aggressive PC in humans, placing it as a potential prognostic marker of aggressive PC.

Modelling Allee effects in a transgenic mosquito population during range expansion

ABSTRACTMosquitoes are vectors for many diseases that cause significant mortality and morbidity. As mosquito populations expand their range, they may undergo mate-finding Allee effects such that their ability to successfully reproduce becomes difficult at low population density. With new technology, creating target specific gene modification may be a viable method for mosquito population control. We develop a mathematical model to investigate the effects of releasing transgenic mosquitoes into newly established, low-density mosquito populations. Our model consists of two life stages (aquatic and adults), which are divided into three genetically distinct groups: heterogeneous and homogeneous transgenic that cause female infertility and a homogeneous wild type. We perform analytical and numerical analyses on the equilibria to determine the level of saturation needed to eliminate mosquitoes in a given area. This model demonstrates the potential for a gene drive system to reduce the spread of invading mosquito populations.

Influence of intergenotypic competition on multigenerational persistence of abiotic stress resistance transgenes in populations of Arabidopsis thaliana.

Reducing crop losses due to abiotic stresses is a major target of agricultural biotechnology that will increase with climate change and global population growth. Concerns, however, have been raised about potential ecological impacts if transgenes become established in wild populations and cause increased competitiveness of weedy or invasive species. Potential risks will be a function of transgene movement, population sizes, and fitness effects on the recipient population. While key components influencing gene flow have been extensively investigated, there have been few studies on factors subsequent to transgene movement that can influence persistence and competitiveness. Here, we performed multiyear, multigenerational, assessment to examine fitness effects and persistence of three mechanistically different abiotic stress tolerance genes: C‐repeat binding factor 3/drought responsive element binding factor 1a (CBF3/DREB1a); Salt overly sensitive 1 (SOS1); and Mannose‐6‐phosphate reductase (M6PR). Transgenic Arabidopsis thaliana overexpressing these genes were grown in pure populations and in competition with wild‐type (WT) parents for six generations spanning a range of field environment conditions. Growth, development, biomass, seed production, and transgene frequency were measured at each generation. Seed planted for each generation was obtained from the previous generation as would occur during establishment of a new genotype in the environment. The three transgenes exhibited different fitness effects and followed different establishment trajectories. In comparison with pure populations, CBF3 lines exhibited reduced dry weight, seed yield, and viable seed yield, relative to WT background. In contrast, overexpression of SOS1 and M6PR did not significantly impact productivity measures in pure populations. In competition with WT, negative fitness effects were magnified. Transgene frequencies were significantly reduced for CBF3 and SOS1 while frequencies of M6PR appeared to be subject to genetic drift. These studies demonstrate the importance of fitness effects and intergenotype competition in influencing persistence of transgenes conferring complex traits.

Morphometric Modifications in Canthon quinquemaculatus Castelnau 1840 (Coleoptera: Scarabaeinae): Sublethal Effects of Transgenic Maize?

The effects of transgenic compounds on non-target organisms remain poorly understood, especially in native insect species. Morphological changes (e.g., changes in body size and shape) may reflect possible responses to environmental stressors, like transgenic toxins. The dung beetle Canthon quinquemaculatus (Coleoptera: Scarabaeinae) is a non-target species found in transgenic crops. We evaluated whether C. quinquemaculatus individuals inhabiting corn fields cultivated with different seed types (conventional, creole and transgenic) present modifications in body shape compared to individuals inhabiting adjacent native forest fragments. We collected C. quinquemaculatus specimens across an agricultural landscape in southern Brazil, during the summer of 2015. Six populations were sampled: three maize crop populations each under a different seed type, and three populations of adjacent forests. After sampling, specimens were subjected to morphometric analyses to discover differences in body shape. We chose fifteen landmarks to describe body shape, and morphometric data were tested with Procrustes ANOVA and Discriminant Analysis. We found that body shape did not differ between individuals collected in conventional and creole crops with their respective adjacent forests (p > 0.05); however, transgenic crop populations differed significantly from those collected in adjacent forests (p < 0.05). Insects in transgenic maize are more oval and have a retraction in the abdominal region, compared with the respective adjacent forest, this result shows the possible effect of transgenic crops on non-target species. This may have implications for the ecosystem service of organic matter removal, carried out by these organisms.

Characterization of novel microneme adhesive repeats (MAR) in Eimeria tenella

BackgroundThe phylum Apicomplexa comprises a wide variety of parasites of significant medical and economic relevance. These parasites have extremely different host and tissue tropisms; for example Toxoplasma gondii can invade virtually any nucleated cell and infect almost all warm-blooded vertebrates, whereas Eimeria tenella infects only chickens and is restricted in its growth to epithelial cells of the caecum. Proteins released from the microneme secretory organelles (MICs) are critical for apicomplexan invasion of host cells and allow parasites to bind a diverse range of host cell oligosaccharide epitopes. MICs bear modular arrangements of sequences with adhesive proteins and interestingly the sialic-acid binding MAR (microneme adhesive repeat) domain containing proteins (MCPs) are suggested to make significant contributions to the different host and tissue tropisms of T. gondii and E. tenella.ResultsIn this study, we evaluated the binding capacity of Type I MAR domains from novel E. tenella MCPs. Variants of the previously described HxT motif were analysed showing that HxT and VxT variants bind, whereas HxS and YxE variants did not. One of these MCP containing a single MAR (EtMCP2) showed an apical localization when expressed as a fusion with the fluorescent reporter mCherry in transgenic populations and a similar pattern of transcripts per zoite during endogenous development in vitro as the well-characterised microneme protein EtMIC2.ConclusionsVariation in the binding properties of the MAR of different EtMCPs was confirmed and their ability to bind a wider range of sialic acids and terminal linkages should be studied. In addition, transgenesis technology has been used for first time in Eimeria parasites as a rapid tool for the study of endogenous protein localization by fusion with a fluorescent reporter.

A High-Throughput Standard PCR-Based Genotyping Method for Determining Transgene Zygosity in Segregating Plant Populations.

In crop research programs that implement transgene-based strategies for trait improvement it is necessary to distinguish between transgene homozygous and hemizygous individuals in segregating populations. Direct methods for determining transgene zygosity are technically challenging, expensive, and require specialized equipment. In this report, we describe a standard PCR-based protocol coupled with capillary electrophoresis that can identify transgene homozygous and hemizygous individuals in a segregating population without knowledge of transgene insertion site. PCR primers were designed to amplify conserved T-DNA segments of the 35S promoter, OCS terminator, and NPTII kanamycin resistance gene in the pHellsgate-8 RNAi construct for the Gossypium hirsutum phytochrome A1 gene. Using an optimized multiplexed reaction mixture and an amplification program of only 10 cycles we could discriminate between transgene homozygous and hemizygous cotton control DNA samples based on PCR product peak characteristics gathered by capillary electrophoresis. The protocol was refined by evaluating segregating transgenic progeny from nine BC1S1 populations derived from crosses between the transgenic cotton parent ‘E-1-7-6’ and other cotton cultivars. OCS PCR product peak height and peak area, normalized by amplification of the native cotton gene GhUBC1, revealed clear bimodal distributions of OCS product characteristics for each BC1S1 population indicating the presence of homozygous and hemizygous clusters which was further confirmed via K-means clustering. BC1S1 plants identified as homozygous or hemizygous were self-fertilized to produce BC1S2 progeny. For the homozygous class, 19/20 BC1S2 families confirmed the homozygous BC1S1 prediction while 21/21 BC1S2 families confirmed the hemizygous prediction of the original parent. This relatively simple protocol provides a reliable, rapid, and high-throughput way of evaluating segregating transgenic populations using methods and equipment common to crop molecular breeding labs.

Eimeria tenella protein trafficking: differential regulation of secretion versus surface tethering during the life cycle

Eimeria spp. are intracellular parasites that have a major impact on poultry. Effective live vaccines are available and the development of reverse genetic technologies has raised the prospect of using Eimeria spp. as recombinant vectors to express additional immunoprotective antigens. To study the ability of Eimeria to secrete foreign antigens or display them on the surface of the sporozoite, transiently transfected populations of E. tenella expressing the fluorescent protein mCherry, linked to endogenous signal peptide (SP) and glycophosphatidylinositol-anchor (GPI) sequences, were examined. The SP from microneme protein EtMIC2 (SP2) allowed efficient trafficking of mCherry to cytoplasmic vesicles and following the C-terminal addition of a GPI-anchor (from surface antigen EtSAG1) mCherry was expressed on the sporozoite surface. In stable transgenic populations, mCherry fused to SP2 was secreted into the sporocyst cavity of the oocysts and after excystation, secretion was detected in culture supernatants but not into the parasitophorous vacuole after invasion. When the GPI was incorporated, mCherry was observed on the sporozites surface and in the supernatant of invading sporozoites. The proven secretion and surface exposure of mCherry suggests that antigen fusions with SP2 and GPI of EtSAG1 may be promising candidates to examine induction of protective immunity against heterologous pathogens.

Nanos-Driven expression of piggyBac transposase induces mobilization of a synthetic autonomous transposon in the malaria vector mosquito, Anopheles stephensi

Transposons are a class of selfish DNA elements that can mobilize within a genome. If mobilization is accompanied by an increase in copy number (replicative transposition), the transposon may sweep through a population until it is fixed in all of its interbreeding members. This introgression has been proposed as the basis for drive systems to move genes with desirable phenotypes into target species. One such application would be to use them to move a gene conferring resistance to malaria parasites throughout a population of vector mosquitos. We assessed the feasibility of using the piggyBac transposon as a gene-drive mechanism to distribute anti-malarial transgenes in populations of the malaria vector, Anopheles stephensi. We designed synthetic gene constructs that express the piggyBac transposase in the female germline using the control DNA of the An. stephensi nanos orthologous gene linked to marker genes to monitor inheritance. Two remobilization events were observed with a frequency of one every 23 generations, a rate far below what would be useful to drive anti-pathogen transgenes into wild mosquito populations. We discuss the possibility of optimizing this system and the impetus to do so.

Transient expression in Nicotiana benthamiana for rapid functional analysis of genes involved in non-photochemical quenching and carotenoid biosynthesis.

SummaryPlants must switch rapidly between light harvesting and photoprotection in response to environmental fluctuations in light intensity. This switch can lead to losses in absorbed energy usage, as photoprotective energy dissipation mechanisms can take minutes to hours to fully relax. One possible way to improve photosynthesis is to engineer these energy dissipation mechanisms (measured as non‐photochemical quenching of chlorophyll a fluorescence, NPQ) to induce and relax more quickly, resulting in smaller losses under dynamic light conditions. Previous studies aimed at understanding the enzymes involved in the regulation of NPQ have relied primarily on labor‐intensive and time‐consuming generation of stable transgenic lines and mutant populations – approaches limited to organisms amenable to genetic manipulation and mapping. To enable rapid functional testing of NPQ‐related genes from diverse organisms, we performed Agrobacterium tumefaciens‐mediated transient expression assays in Nicotiana benthamiana to test if NPQ kinetics could be modified in fully expanded leaves. By expressing Arabidopsis thaliana genes known to be involved in NPQ, we confirmed the viability of this method for studying dynamic photosynthetic processes. Subsequently, we used naturally occurring variation in photosystem II subunit S, a modulator of NPQ in plants, to explore how differences in amino acid sequence affect NPQ capacity and kinetics. Finally, we functionally characterized four predicted carotenoid biosynthesis genes from the marine algae Nannochloropsis oceanica and Thalassiosira pseudonana and examined the effect of their expression on NPQ in N. benthamiana. This method offers a powerful alternative to traditional gene characterization methods by providing a fast and easy platform for assessing gene function in planta.

Identifikasi Perubahan Karakter Agronomis Padi Transgenik Penanda Aktivasi cv. Asemandi Generasi T1

&lt;p&gt;The activation-tagged populations of transgenic rice cv.&lt;br /&gt;Asemandi have been developed by introducing construct of&lt;br /&gt;activation taq Ac/Ds into rice genom of cv. Asemandi. The T1&lt;br /&gt;transgenic rice populations cv. Asemandi containing&lt;br /&gt;construct of activation taq have been obtained and needed&lt;br /&gt;to be characterized. This study aimed to identify the Basta&lt;br /&gt;herbicide resistant plants, transgenic plants containing hpt&lt;br /&gt;and bar genes, and changes in agronomic traits. Basta&lt;br /&gt;resistant plant was identified by treating leaf with basta&lt;br /&gt;solution. Hpt and bar genes were detected by PCR using&lt;br /&gt;specific primers. Phenotype characters were identified by&lt;br /&gt;observing and measuring their agronomic parameters. The&lt;br /&gt;study results showed that out of 315 rice transgenic cv.&lt;br /&gt;Asemandi T1 treated with Basta solution, 176 (55.87%) plants&lt;br /&gt;were indicated to be resistant to Basta. The results of PCR&lt;br /&gt;analysis revealed that eight rice transgenic cv. Asemandi&lt;br /&gt;tested contained both hpt and bar genes. In general,&lt;br /&gt;compared to the nontransgenic plants, there were changes&lt;br /&gt;in several agronomic parameters of T1 transgenic plants cv.&lt;br /&gt;Asemandi, including plant height, days to flowering, days to&lt;br /&gt;harvesting, periods of grain filling, and weight of 100 grains.&lt;br /&gt;Correlation analysis showed that there was no correlation&lt;br /&gt;between days of harvesting to weight of 100 grains in&lt;br /&gt;transgenic rice, but there was correlation in nontransgenic&lt;br /&gt;rice. Transgenic rice plants cv. Asemandi with changes in&lt;br /&gt;the agronomic characters will be useful for further study,&lt;br /&gt;such as to analyze the function of the genes.&lt;/p&gt;

Viral proteins expressed in the protozoan parasite Eimeria tenella are detected by the chicken immune system.

BackgroundEimeria species are parasitic protozoa that cause coccidiosis, an intestinal disease commonly characterised by malabsorption, diarrhoea and haemorrhage that is particularly important in chickens. Vaccination against chicken coccidiosis is effective using wild-type or attenuated live parasite lines. The development of protocols to express foreign proteins in Eimeria species has opened up the possibility of using Eimeria live vaccines to deliver heterologous antigens and function as multivalent vaccine vectors that could protect chickens against a range of pathogens.ResultsIn this study, genetic complementation was used to express immunoprotective virus antigens in Eimeria tenella. Infectious bursal disease virus (IBDV) causes Gumboro, an immunosuppressive disease that affects productivity and can interfere with the efficacy of poultry vaccination programmes. Infectious laryngotracheitis virus (ILTV) causes a highly transmissible respiratory disease for which strong cellular immunity and antibody responses are required for effective vaccination. Genes encoding the VP2 protein from a very virulent strain of IBDV (vvVP2) and glycoprotein I from ILTV (gI) were cloned downstream of 5’Et-Actin or 5’Et-TIF promoter regions in plasmids that also contained a mCitrine fluorescent reporter cassette under control of the 5’Et-MIC1 promoter. The plasmids were introduced by nucleofection into E. tenella sporozoites, which were then used to infect chickens. Progeny oocysts were sorted by FACS and passaged several times in vivo until the proportion of fluorescent parasites in each transgenic population reached ~20 % and the number of transgene copies per parasite genome decreased to < 10. All populations were found to transcribe and express the transgene and induced the generation of low titre, transgene-specific antibodies when used to immunise chickens.ConclusionsE. tenella can express antigens of other poultry pathogens that are successfully recognised by the chicken immune system. Nonetheless, further work has to be done in order to improve the levels of expression for its future use as a multivalent vaccine vector.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1756-2) contains supplementary material, which is available to authorized users.