Transgenic Camelina Research Articles

Получение и анализ безмаркерных масличных растений Camelina sativa (L.), экспрессирующих ген антимикробного пептида цекропина Р1

The marker-free transgenic Camelina sativa (L.) plants carrying a synthetic gene for cecropin P1, an antimicrobial peptide, under the control of the cauliflower mosaic virus 35S RNA promoter have been obtained and analyzed. The plants were transformed by the agrobacterial binary vector free of selection genes of antibiotic and herbicide resistance. Screening of the marker-free transformants was performed by measuring the antibacterial activity of cecropin P1 and by enzyme immunoassay. The obtained plants exhibited increased resistance to the infection by the Erwinia carotovora bacteria, Fusarium graminearum fungi, and to oxidative stress during infection. The analysis of the fatty acid composition of seed oil showed an increased amount of α-linolenic acid in the transgenic camelina lines as compared to unmodified plants. The obtained results indicate that the gene for cecropin P1 can be included in the integral antistress plant protective system. Camelina sativa, cecropin Р1, agrobacterium-mediated transformation, marker-free transgenic plants, fatty acid compositions, microbial phytopathogenes, oxidative stress, resistance, cecropin P1. The work was financially supported by State job № 0101-2014-0046, № РК 01201352439 and with the partial financial support the Russian Fund for Basic Investigation, Grants № 18-08-00752, № 16-04-00623.

Production of Transgenic <i>Camelina sativa</i> Plants via <i>Agrobacterium</i>-Mediated Transformation of Shoot Apical Meristems

A method to produce transgenic Camelina sativa plants in cvs. PI650159 and PI650161 was developed. Micropropagated shoot meristem cultures were established from in vitro germinated seedlings and used as target tissues for Agrobacterium-mediated transformation. A plasmid harboring enhanced green fluorescent protein, β glucuronidase and neomycin phosphotransferase II genes were used to optimize parameters for transgenic plant production. Kanamycin at 40 mg·l-1 was effective in suppression of non-transformed cells while permitting growth of transgenic tissues. Shoot apical meristems co-cultivated with Agrobacterium exhibited stable enhanced green fluorescence protein (EGFP) and β glucuronidase (GUS) expression after culture on plant regeneration medium. We observed transformation efficiencies of 53.33% in cv. PI650159 and 98.33% in cv. PI650161. The presence of transgenes in both cultivars was confirmed by PCR, while quantitative real-time PCR detected single copy integration in Pl650161 and two copy integration in Pl650159. Transgenic plants exhibited EGFP and GUS expression in all tissues including shoots, leaves, buds, floral organs, seeds, and pods. Our results demonstrate a simple and efficient technique using apical shoot meristems for production of transgenic C. sativa plants that can be used for transfer of desirable traits.

Comparative transcriptome and metabolome analysis suggests bottlenecks that limit seed and oil yields in transgenic Camelina sativa expressing diacylglycerol acyltransferase 1 and glycerol-3-phosphate dehydrogenase

BackgroundCamelina sativa has attracted much interest as alternative renewable resources for biodiesel, other oil-based industrial products and a source for edible oils. Its unique oil attributes attract research to engineering new varieties of improved oil quantity and quality. The overexpression of enzymes catalyzing the synthesis of the glycerol backbone and the sequential conjugation of fatty acids into this backbone is a promising approach for increasing the levels of triacylglycerol (TAG). In a previous study, we co-expressed the diacylglycerol acyltransferase (DGAT1) and glycerol-3-phosphate dehydrogenase (GPD1), involved in TAG metabolism, in Camelina seeds. Transgenic plants exhibited a higher-percentage seed oil content, a greater seed mass, and overall improved seed and oil yields relative to wild-type plants. To further increase seed oil content in Camelina, we utilized metabolite profiling, in conjunction with transcriptome profiling during seed development to examine potential rate-limiting step(s) in the production of building blocks for TAG biosynthesis.ResultsTranscriptomic analysis revealed approximately 2518 and 3136 transcripts differentially regulated at significant levels in DGAT1 and GPD1 transgenics, respectively. These transcripts were found to be involved in various functional categories, including alternative metabolic routes in fatty acid synthesis, TAG assembly, and TAG degradation. We quantified the relative contents of over 240 metabolites. Our results indicate major metabolic switches in transgenic seeds associated with significant changes in the levels of glycerolipids, amino acids, sugars, and organic acids, especially the TCA cycle and glycolysis intermediates.ConclusionsFrom the transcriptomic and metabolomic analysis of DGAT1, GPD1 and DGAT1 + GPD1 expressing lines of C. sativa, we conclude that TAG production is limited by (1) utilization of fixed carbon from the source tissues supported by the increase in glycolysis pathway metabolites and decreased transcripts levels of transcription factors controlling fatty acids synthesis; (2) TAG accumulation is limited by the activity of lipases/hydrolases that hydrolyze TAG pool supported by the increase in free fatty acids and monoacylglycerols. This comparative transcriptomics and metabolomics approach is useful in understanding the regulation of TAG biosynthesis, identifying bottlenecks, and the corresponding genes controlling these pathways identified as limitations, for generating Camelina varieties with improved seed and oil yields.

Towards the synthetic design of camelina oil enriched in tailored acetyl-triacylglycerols with medium-chain fatty acids.

The ability to manipulate expression of key biosynthetic enzymes has allowed the development of genetically modified plants that synthesise unusual lipids that are useful for biofuel and industrial applications. By taking advantage of the unique activities of enzymes from different species, tailored lipids with a targeted structure can be conceived. In this study we demonstrate the successful implementation of such an approach by metabolically engineering the oilseed crop Camelina sativa to produce 3-acetyl-1,2-diacyl-sn-glycerols (acetyl-TAGs) with medium-chain fatty acids (MCFAs). Different transgenic camelina lines that had been genetically modified to produce MCFAs through the expression of MCFA-specific thioesterases and acyltransferases were retransformed with the Euonymus alatus gene for diacylglycerol acetyltransferase (EaDAcT) that synthesises acetyl-TAGs. Concomitant RNAi suppression of acyl-CoA:diacylglycerol acyltransferase increased the levels of acetyl-TAG, with up to 77 mole percent in the best lines. However, the total oil content was reduced. Analysis of the composition of the acetyl-TAG molecular species using electrospray ionisation mass spectrometry demonstrated the successful synthesis of acetyl-TAG containing MCFAs. Field growth of high-yielding plants generated enough oil for quantification of viscosity. As part of an ongoing design-test-learn cycle, these results, which include not only the synthesis of 'designer' lipids but also their functional analysis, will lead to the future production of such molecules tailored for specific applications.

A transgene design for enhancing oil content in Arabidopsis and Camelina seeds

BackgroundIncreasing the oil yield is a major objective for oilseed crop improvement. Oil biosynthesis and accumulation are influenced by multiple genes involved in embryo and seed development. The leafy cotyledon1 (LEC1) is a master regulator of embryo development that also enhances the expression of genes involved in fatty acid biosynthesis. We speculated that seed oil could be increased by targeted overexpression of a master regulating transcription factor for oil biosynthesis, using a downstream promoter for a gene in the oil biosynthesis pathway. To verify the effect of such a combination on seed oil content, we made constructs with maize (Zea mays) ZmLEC1 driven by serine carboxypeptidase-like (SCPL17) and acyl carrier protein (ACP5) promoters, respectively, for expression in transgenic Arabidopsis thaliana and Camelina sativa.ResultsAgrobacterium-mediated transformation successfully generated Arabidopsis and Camelina lines that overexpressed ZmLEC1 under the control of a seed-specific promoter. This overexpression does not appear to be detrimental to seed vigor under laboratory conditions and did not cause observable abnormal growth phenotypes throughout the life cycle of the plants. Overexpression of ZmLEC1 increased the oil content in mature seeds by more than 20% in Arabidopsis and 26% in Camelina.ConclusionThe findings suggested that the maize master regulator, ZmLEC1, driven by a downstream seed-specific promoter, can be used to increase oil production in Arabidopsis and Camelina and might be a promising target for increasing oil yield in oilseed crops.0

Identification of bottlenecks in the accumulation of cyclic fatty acids in camelina seed oil.

SummaryModified fatty acids (mFA) have diverse uses; for example, cyclopropane fatty acids (CPA) are feedstocks for producing coatings, lubricants, plastics and cosmetics. The expression of mFA‐producing enzymes in crop and model plants generally results in lower levels of mFA accumulation than in their natural‐occurring source plants. Thus, to further our understanding of metabolic bottlenecks that limit mFA accumulation, we generated transgenic Camelina sativa lines co‐expressing Escherichia coli cyclopropane synthase (EcCPS) and Sterculia foetida lysophosphatidic acid acyltransferase (SfLPAT). In contrast to transgenic CPA‐accumulating Arabidopsis, CPA accumulation in camelina caused only minor changes in seed weight, germination rate, oil accumulation and seedling development. CPA accumulated to much higher levels in membrane than storage lipids, comprising more than 60% of total fatty acid in both phosphatidylcholine (PC) and phosphatidylethanolamine (PE) versus 26% in diacylglycerol (DAG) and 12% in triacylglycerol (TAG) indicating bottlenecks in the transfer of CPA from PC to DAG and from DAG to TAG. Upon co‐expression of SfLPAT with EcCPS, di‐CPA‐PC increased by ~50% relative to lines expressing EcCPS alone with the di‐CPA‐PC primarily observed in the embryonic axis and mono‐CPA‐PC primarily in cotyledon tissue. EcCPS‐SfLPAT lines revealed a redistribution of CPA from the sn‐1 to sn‐2 positions within PC and PE that was associated with a doubling of CPA accumulation in both DAG and TAG. The identification of metabolic bottlenecks in acyl transfer between site of synthesis (phospholipids) and deposition in storage oils (TAGs) lays the foundation for the optimizing CPA accumulation through directed engineering of oil synthesis in target crops.

Tailoring seed oil composition in the real world: optimising omega-3 long chain polyunsaturated fatty acid accumulation in transgenic Camelina sativa

There is considerable interest in the de novo production of omega-3 long chain polyunsaturated fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), not least of all given the importance of these fatty acids in both aquaculture and human nutrition. Previously we have demonstrated the feasibility of using metabolic engineering in transgenic plants (Camelina sativa) to modify the seed oil composition to now include EPA and/or DHA. In this study, we further tailored the seed oil profile to reduce the omega-6 content, and evaluated the performance of such GM plants under field conditions (i.e. environmental releases), in terms of agronomic performance and also the lipidomic profile of seed oil. We used MALDI- mass spectrometry imaging to identify discrete tissue-types in the seed in which these non-native fatty acids preferentially accumulated. Collectively, these data provide new insights into the complexity of plant lipid metabolism and the challenges associated with predictive manipulation of these pathways. However, this study identified the likely dispensable nature of a Δ12-desturase activity in our omega-3 metabolic engineering rationales for Camelina.

Erratum to: Replacement of Marine Fish Oil with de novo Omega-3 Oils from Transgenic Camelina sativa in Feeds for Gilthead Sea Bream (Sparus aurata L.).

Omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA) are essential components of the diet of all vertebrates. The major dietary source of n-3 LC-PUFA for humans has been fish and seafood but, paradoxically, farmed fish are also reliant on marine fisheries for fish meal and fish oil (FO), traditionally major ingredients of aquafeeds. Currently, the only sustainable alternatives to FO are vegetable oils, which are rich in C18 PUFA, but devoid of the eicosapentaenoic (EPA) and docosahexaenoic acids (DHA) abundant in FO. Two new n-3 LC-PUFA sources obtained from genetically modified (GM) Camelina sativa containing either EPA alone (ECO) or EPA and DHA (DCO) were compared to FO and wild-type camelina oil (WCO) in juvenile sea bream. Neither ECO nor DCO had any detrimental effects on fish performance, although final weight of ECO-fed fish (117 g) was slightly lower than that of FO- and DCO-fed fish (130 and 127 g, respectively). Inclusion of the GM-derived oils enhanced the n-3 LC-PUFA content in fish tissues compared to WCO, although limited biosynthesis was observed indicating accumulation of dietary fatty acids. The expression of genes involved in several lipid metabolic processes, as well as fish health and immune response, in both liver and anterior intestine were altered in fish fed the GM-derived oils. This showed a similar pattern to that observed in WCO-fed fish reflecting the hybrid fatty acid profile of the new oils. Overall the data indicated that the GM-derived oils could be suitable alternatives to dietary FO in sea bream.

An oil containing EPA and DHA from transgenic Camelina sativa to replace marine fish oil in feeds for Atlantic salmon (Salmo salar L.): Effects on intestinal transcriptome, histology, tissue fatty acid profiles and plasma biochemistry

New de novo sources of omega 3 (n-3) long chain polyunsaturated fatty acids (LC-PUFA) are required as alternatives to fish oil in aquafeeds in order to maintain adequate levels of the beneficial fatty acids, eicosapentaenoic and docosahexaenoic (EPA and DHA, respectively). The present study investigated the use of an EPA+DHA oil derived from transgenic Camelina sativa in Atlantic salmon (Salmo salar) feeds containing low levels of fishmeal (35%) and fish oil (10%), reflecting current commercial formulations, to determine the impacts on tissue fatty acid profile, intestinal transcriptome, and health of farmed salmon. Post-smolt Atlantic salmon were fed for 12-weeks with one of three experimental diets containing either a blend of fish oil/rapeseed oil (FO), wild-type camelina oil (WCO) or transgenic camelina oil (DCO) as added lipid source. The DCO diet did not affect any of the fish performance or health parameters studied. Analyses of the mid and hindgut transcriptomes showed only mild effects on metabolism. Flesh of fish fed the DCO diet accumulated almost double the amount of n-3 LC-PUFA than fish fed the FO or WCO diets, indicating that these oils from transgenic oilseeds offer the opportunity to increase the n-3 LC-PUFA in farmed fish to levels comparable to those found a decade ago.

An update to the Canadian range, abundance, and ploidy of Camelina spp. (Brassicaceae) east of the Rocky Mountains

The distribution and abundance of three Camelina species introduced to Canada is unknown, but critical for evaluating the risks associated with unconfined release of transgenic Camelina sativa (L.) Crantz (2n = 40). Furthermore, previous reports suggest Canadian populations of Camelina microcarpa Andrz. ex DC. vary for ploidy and ability to hybridize with C. sativa. We completed 8 weeks of field work in Alberta, Saskatchewan, Manitoba, southern Ontario, Quebec, and the Maritimes. We determined the ploidy composition of the populations found. We did not locate Camelina alyssum (Mill.) Thell., but located four sites with C. sativa and 34 with C. microcarpa. Eleven C. microcarpa populations were tetraploid (2n = 26, 1.00pg/2C) and 22 were hexaploid (2n = 40, 1.50pg/2C), while two populations were mixed. We examined material from botanical gardens and plant gene resource centres assessing total nuclear DNA content and completing chromosome counts for each species and cytotype identified, to determine whether tetraploid and hexaploid C. microcarpa were included in these collections. No tetraploid material was included in the C. microcarpa accessions received; however, a diploid (2n = 12, 0.54pg/2C) was found. Given the current geographic ranges, abundance, and chromosome counts of these species, the greatest risk of hybridization with transgenic C. sativa is from hexaploid C. microcarpa.

Accumulation of medium-chain, saturated fatty acyl moieties in seed oils of transgenic Camelina sativa.

With its high seed oil content, the mustard family plant Camelina sativa has gained attention as a potential biofuel source. As a bioenergy crop, camelina has many advantages. It grows on marginal land with low demand for water and fertilizer, has a relatively short life cycle, and is stress tolerant. As most other crop seed oils, camelina seed triacylglycerols (TAGs) consist of mostly long, unsaturated fatty acyl moieties, which is not desirable for biofuel processing. In our efforts to produce shorter, saturated chain fatty acyl moieties in camelina seed oil for conversion to jet fuel, a 12:0-acyl-carrier thioesterase gene, UcFATB1, from California bay (Umbellularia californica Nutt.) was expressed in camelina seeds. Up to 40% of short chain laurate (C12:0) and myristate (C14:0) were present in TAGs of the seed oil of the transgenics. The total oil content and germination rate of the transgenic seeds were not affected. Analysis of positions of these two fatty acyl moieties in TAGs indicated that they were present at the sn-1 and sn-3 positions, but not sn-2, on the TAGs. Suppression of the camelina KASII genes by RNAi constructs led to higher accumulation of palmitate (C16:0), from 7.5% up to 28.5%, and further reduction of longer, unsaturated fatty acids in seed TAGs. Co-transformation of camelina with both constructs resulted in enhanced accumulation of all three medium-chain, saturated fatty acids in camelina seed oils. Our results show that a California bay gene can be successfully used to modify the oil composition in camelina seed and present a new biological alternative for jet fuel production.

Tailoring the composition of novel wax esters in the seeds of transgenic Camelina sativa through systematic metabolic engineering.

SummaryThe functional characterization of wax biosynthetic enzymes in transgenic plants has opened the possibility of producing tailored wax esters (WEs) in the seeds of a suitable host crop. In this study, in addition to systematically evaluating a panel of WE biosynthetic activities, we have also modulated the acyl‐CoA substrate pool, through the co‐expression of acyl‐ACP thioesterases, to direct the accumulation of medium‐chain fatty acids. Using this combinatorial approach, we determined the additive contribution of both the varied acyl‐CoA pool and biosynthetic enzyme substrate specificity to the accumulation of non‐native WEs in the seeds of transgenic Camelina plants. A total of fourteen constructs were prepared containing selected FAR and WS genes in combination with an acyl‐ACP thioesterase. All enzyme combinations led to the successful production of wax esters, of differing compositions. The impact of acyl‐CoA thioesterase expression on wax ester accumulation varied depending on the substrate specificity of the WS. Hence, co‐expression of acyl‐ACP thioesterases with Marinobacter hydrocarbonoclasticus WS and Marinobacter aquaeolei FAR resulted in the production of WEs with reduced chain lengths, whereas the co‐expression of the same acyl‐ACP thioesterases in combination with Mus musculus WS and M. aquaeolei FAR had little impact on the overall final wax composition. This was despite substantial remodelling of the acyl‐CoA pool, suggesting that these substrates were not efficiently incorporated into WEs. These results indicate that modification of the substrate pool requires careful selection of the WS and FAR activities for the successful high accumulation of these novel wax ester species in Camelina seeds.

Inoculation of Soil with Plant Growth Promoting Bacteria Producing 1-Aminocyclopropane-1-Carboxylate Deaminase or Expression of the Corresponding acdS Gene in Transgenic Plants Increases Salinity Tolerance in Camelina sativa.

Camelina sativa (camelina) is an oilseed crop touted for use on marginal lands; however, it is no more tolerant of soil salinity than traditional crops, such as canola. Plant growth-promoting bacteria (PGPB) that produce 1-aminocyclopropane-1-carboxylate deaminase (ACC deaminase) facilitate plant growth in the presence of abiotic stresses by reducing stress ethylene. Rhizospheric and endophytic PGPB and the corresponding acdS- mutants of the latter were examined for their ability to enhance tolerance to salt in camelina. Stimulation of growth and tolerance to salt was correlated with ACC deaminase production. Inoculation of soil with wild-type PGPB led to increased shoot length in the absence of salt, and increased seed production by approximately 30–50% under moderately saline conditions. The effect of ACC deaminase was further examined in transgenic camelina expressing a bacterial gene encoding ACC deaminase (acdS) under the regulation of the CaMV 35S promoter or the root-specific rolD promoter. Lines expressing acdS, in particular those using the rolD promoter, showed less decline in root length and weight, increased seed production, better seed quality and higher levels of seed oil production under salt stress. This study clearly demonstrates the potential benefit of using either PGPB that produce ACC deaminase or transgenic plants expressing the acdS gene under the control of a root-specific promoter to facilitate plant growth, seed production and seed quality on land that is not normally suitable for the majority of crops due to high salt content.

Proteome rebalancing in transgenic Camelina occurs within the enlarged proteome induced by β-carotene accumulation and storage protein suppression

Oilseed crops are global commodities for their oil and protein seed content. We have engineered the oilseed Camelina sativa to exhibit increased protein content with a slight decrease in oil content. The introduction of a phytoene synthase gene with an RNAi cassette directed to suppress the storage protein 2S albumin resulted in seeds with an 11-24% elevation in overall protein. The phytoene synthase cassette alone produced enhanced β-carotene content of an average 275±6.10μg/g dry seed and an overall altered seed composition of 11% less protein and comparable nontransgenic amounts of both oil and carbohydrates. Stacking an RNAi to suppress the major 2S storage protein resulted in seeds that contain elevated protein and slight decrease in oil and carbohydrate amounts showing that Camelina rebalances its proteome within an enlarged protein content genotype. In both β-carotene enhanced seeds with/without RNAi2S suppression, the seed size was noticeably enlarged compared to nontransgenic counterpart seeds. Metabolic analysis of maturing seeds indicate that the enhanced β-carotene trait had the larger effect than the RNAi2S suppression on the seed metabolome. The use of a GRAS (generally regarded as safe) β-carotene as a visual marker in a floral dip transformation system, such as Camelina, might eliminate the need for costly regulatory and controversial antibiotic resistance markers. β-carotene enhanced RNAi2S suppressed Camelina seeds could be further developed as a rapid heterologous protein production platform in a nonfood crop leveraging its enlarged protein content and visual marker.

Nutritional Evaluation of an EPA-DHA Oil from Transgenic Camelina sativa in Feeds for Post-Smolt Atlantic Salmon (Salmo salar L.).

Vegetable oils (VO) are possible substitutes for fish oil in aquafeeds but their use is limited by their lack of omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA). However, oilseed crops can be modified to produce n-3 LC-PUFA such as eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, representing a potential option to fill the gap between supply and demand of these important nutrients. Camelina sativa was metabolically engineered to produce a seed oil with around 15% total n-3 LC-PUFA to potentially substitute for fish oil in salmon feeds. Post-smolt Atlantic salmon (Salmo salar) were fed for 11-weeks with one of three experimental diets containing either fish oil (FO), wild-type Camelina oil (WCO) or transgenic Camelina oil (DCO) as added lipid source to evaluate fish performance, nutrient digestibility, tissue n-3 LC-PUFA, and metabolic impact determined by liver transcriptome analysis. The DCO diet did not affect any of the performance or health parameters studied and enhanced apparent digestibility of EPA and DHA compared to the WCO diet. The level of total n-3 LC-PUFA was higher in all the tissues of DCO-fed fish than in WCO-fed fish with levels in liver similar to those in fish fed FO. Endogenous LC-PUFA biosynthetic activity was observed in fish fed both the Camelina oil diets as indicated by the liver transcriptome and levels of intermediate metabolites such as docosapentaenoic acid, with data suggesting that the dietary combination of EPA and DHA inhibited desaturation and elongation activities. Expression of genes involved in phospholipid and triacylglycerol metabolism followed a similar pattern in fish fed DCO and WCO despite the difference in n-3 LC-PUFA contents.

Engineering synthetic pathways for the production of pharmaceutical sciadonic acid in transgenic oilseed Camelina sativa

Engineering synthetic pathways for the production of pharmaceutical sciadonic acid in transgenic oilseed Camelina sativa

Functional analysis of diacylglycerol acyltransferase1 genes from Camelina sativa and effects of CsDGAT1B overexpression on seed mass and storage oil content in C. sativa

Camelina (Camelina sativa), which belongs to the Brassicaeae family, is an emerging oilseed crop with the potential to expand biodiesel production to arid land. During storage oil synthesis, diacylglycerol acyltrasferase1 (DGAT1) catalyzes the conversion of diacylglycerol (DAG) and free fatty acids to triacylglycerol (TAG). In this study, three DGAT1 genes (CsDGAT1A, CsDGAT1B, and CsDGAT1C) were isolated from developing C. sativa seeds. The deduced amino acid sequences of the three CsDGAT1 genes shared more than 84 % identity with those of DGAT1 genes from Arabidopsis thaliana and Brassica napus. CsDGAT1A, B, and C transcripts were detected in various C. sativa organs, including developing seeds. Fluorescent protein-fused CsDGAT1A, B, and C were localized in the endoplasmic reticulum (ER) of tobacco epidermal cells. When the CsDGAT1A, B, and C genes under the control of the BnNapin promoter were expressed in an Arabidopsis AS11 mutant, which is defective in DGAT1, the amounts and composition of total fatty acids in dry seeds were restored to those of the wild type, indicating the three CsDGAT1 genes to be functionally active. In transgenic C. sativa plants overexpressing CsDGAT1B, the levels of total seed oils were increased by ~24 % compared with non-transgenic lines. Transgenic C. sativa embryos with enhanced seed oil contents harbored larger embryonic cells and a greater number of cells compared with the wild type. Transgenic Camelina plants with increased oil contents can be used as renewable resources for the production of biodiesel and non-petroleum-based biomaterials.

Overexpression of a synthetic insect–plant geranyl pyrophosphate synthase gene in Camelina sativa alters plant growth and terpene biosynthesis

A novel plastidial homodimeric insect-plant geranyl pyrophosphate synthase gene is synthesized from three different cDNA origins. Its overexpression in Camelina sativa effectively alters plant development and terpenoid metabolism. Geranyl pyrophosphate synthase (GPPS) converts one isopentenyl pyrophosphate and dimethylallyl pyrophosphate to GPP. Here, we report a synthetic insect-plant GPPS gene and effects of its overexpression on plant growth and terpenoid metabolism of Camelina sativa. We synthesized a 1353-bp cDNA, namely PTP-MpGPPS. This synthetic cDNA was composed of a 1086-bp cDNA fragment encoding a small GPPS isomer of the aphid Myzus persicae (Mp), 240-bp Arabidopsis thaliana cDNA fragment encoding a plastidial transit peptide (PTP), and a 27-bp short cDNA fragment encoding a human influenza hemagglutinin tag peptide. Structural modeling showed that the deduced protein was a homodimeric prenyltransferase. Confocal microscopy analysis demonstrated that the PTP-MpGPPS fused with green florescent protein was localized in the plastids. The synthetic PTP-MpGPPS cDNA driven by 2×35S promoters was introduced into Camelina (Camelina sativa) by Agrobacterium-mediated transformation and its overexpression in transgenic plants were demonstrated by western blot. T2 and T3 progeny of transgenic plants developed larger leaves, grew more and longer internodes, and flowered earlier than wild-type plants. Metabolic analysis showed that the levels of beta-amyrin and campesterol were higher in tissues of transgenic plants than in those of wild-type plants. Fast isoprene sensor analysis demonstrated that transgenic Camelina plants emitted significantly less isoprene than wild-type plants. In addition, transcriptional analyses revealed that the expression levels of gibberellic acid and brassinosteroids-responsive genes were higher in transgenic plants than in wild-type plants. Taken together, these data demonstrated that this novel synthetic insect-plant GPPS cDNA was effective to improve growth traits and alter terpenoid metabolism of Camelina.

A Transgenic Camelina sativa Seed Oil Effectively Replaces Fish Oil as a Dietary Source of Eicosapentaenoic Acid in Mice

Background: Fish currently supplies only 40% of the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) required to allow all individuals globally to meet the minimum intake recommendation of 500 mg/d. Therefore, alternative sustainable sources are needed.Objective: The main objective was to investigate the ability of genetically engineered Camelina sativa (20% EPA) oil (CO) to enrich tissue EPA and DHA relative to an EPA-rich fish oil (FO) in mammals.Methods: Six-week-old male C57BL/6J mice were fed for 10 wk either a palm oil–containing control (C) diet or diets supplemented with EPA-CO or FO, with the C, low-EPA CO (COL), high-EPA CO (COH), low-EPA FO (FOL), and high-EPA FO (FOH) diets providing 0, 0.4, 3.4, 0.3, and 2.9 g EPA/kg diet, respectively. Liver, muscle, and brain were collected for fatty acid analysis, and blood glucose and serum lipids were quantified. The expression of selected hepatic genes involved in EPA and DHA biosynthesis and in modulating their cellular impact was determined.Results: The oils were well tolerated, with significantly greater weight gain in the COH and FOH groups relative to the C group (P < 0.001). Significantly lower (36–38%) blood glucose concentrations were evident in the FOH and COH mice relative to C mice (P < 0.01). Hepatic EPA concentrations were higher in all EPA groups relative to the C group (P < 0.001), with concentrations of 0.0, 0.4, 2.9, 0.2, and 3.6 g/100 g liver total lipids in the C, COL, COH, FOL, and FOH groups, respectively. Comparable dose-independent enrichments of liver DHA were observed in mice fed CO and FO diets (P < 0.001). Relative to the C group, lower fatty acid desaturase 1 (Fads1) expression (P < 0.005) was observed in the COH and FOH groups. Higher fatty acid desaturase 2 (Fads2), peroxisome proliferator–activated receptor α (Ppara), and peroxisome proliferator–activated receptor γ (Pparg) (P < 0.005) expressions were induced by CO. No impact of treatment on liver X receptor α (Lxra) or sterol regulatory element-binding protein 1c (Srebp1c) was evident.Conclusions: Oil from transgenic Camelina is a bioavailable source of EPA in mice. These data provide support for the future assessment of this oil in a human feeding trial.

Dietary supplements: health from the ocean?

Dietary supplements: health from the ocean?