Efficient and stable transformation of Dunaliella pseudosalina by 3 strains of Agrobacterium tumefaciens

Introduction: Several platforms including mammalian, plant and insect cells as well as bacteria, yeasts, and microalgae are available for the production of recombinant proteins. Low efficiency of delivery systems, extracellular and intracellular degradation of foreign genes during transformation, difficulties in targeting and importing into the nucleus, and finally problems in integration into nuclear genome are the most bottlenecks of classical plasmids for producing recombinant proteins. Owing to high growth rate, no common pathogen with humans, being utilized as humans’ food, and capability to perform N-glycosylation, microalgae are proposed as an ideal system for such biotechnological approaches. Here, Agrobacterium tumefaciens is introduced as an alternative tool for transformation of the microalga Dunaliella pseudosalina. Methods: The transformation of gfp gene into the D. pseudosalina was evaluated by three strains including EHA101, GV3301 and GV3850 of A. tumefaciens. The integrating and expression of gfp gene were determined by PCR, RT-PCR, Q-PCR and SDS-PAGE analyses. Results: The T-DNA of pCAMBIA1304 plasmid was successfully integrated into the genome of the microalgal cells. Although all of the strains were able to transform the algal cells, GV3301 possessed higher potential to transform the microalgal cells in comparison to EHA101 and GV3850 strains. Moreover, the stability of gfp gene was successfully established during a course of two months period in the microalgal genome. Conclusion: Agrobacterium is introduced as a competent system for stable transformation of Dunaliella strains in order to produce eukaryotic recombinant proteins

Growth and antioxidant system responses of maize (Zea mays L.) seedling to different concentration of pyrene in a controlled environment

Polycyclic aromatic hydrocarbons (PAHs) are a class of organic pollutants effecting different aspects of plants physiology. To assess the physiological responses of plants to PAHs, maize (Zea mays) was treated with 25, 50, 75, and 100 ppm of pyrene and after 21 days, the activity of some antioxidant enzymes, malondialdehyde (MDA), total flavonoid, total anthocyanin, and soluble sugar contents were measured in shoots and roots of plants. Pyrene led to increase MDA content as well as CAT, POD, and SOD activities. Increase in pyrene concentration reduced all studied growth variables and significantly increased photosynthetic pigments contents of plants. Soluble sugar content was significantly higher in the shoot, while that was reduced in the roots through increasing of pyrene concentration (p &lt; 0.05). Also, the increase of pyrene concentration decreased total flavonoid content compared to anthocyanin content. In conclusion, these findings supported the hypothesis that pyrene toxicity induces oxidative stress in the maize plant and it also increases the antioxidant systems in order to moderating stress condition. However, the antioxidant system of maize was not strong enough to eliminate all produced ROS at high concentrations, thus this caused oxidative damage to the plant and decreased its growth variables.</p

Designing a new generation of expression toolkits for engineering of green microalgae; robust production of human interleukin-2

Introduction: Attributable to some critical features especially the similarity of the protein synthesis machinery between humans and microalgae, these microorganisms can be utilized for the expression of many recombinant proteins. However, low and unstable gene expression levels prevent the further development of microalgae biotechnology towards protein production. Methods: Here, we designed a novel "Gained Agrobacterium-2A plasmid for microalgae expression" (named GAME plasmid) for the production of the human interleukin-2 using three model microalgae, including Chlamydomonas reinhardtii, Chlorella vulgaris, and Dunaliella salina. The GAME plasmid harbors a native chimeric hsp70/Int-1/rbcS2 promoter, the microalgae specific Kozak sequence, a novel hybrid 2A peptide, and Int-1 and Int-3 of the rbcS2 gene in its expression cassette. Results: The obtained data confirmed that the GAME plasmid can transform the microalgae with high transformation frequency. Molecular and proteomic analyses revealed the stable and robust production of the hIL-2 by the GAME plasmid in the microalgae. According to the densimetric analysis, the microalgae can accumulate the produced protein about 0.94% of the total soluble protein content. The ELISA data confirmed that the produced hIL-2 possesses the same conformation pattern with the acceptable biological activity found naturally in humans. Conclusion: Most therapeutic proteins need post-translational modifications for their correct conformation, biological function, and half-life. Accordingly, microalgae could be considered as a cost-effective and more powerful platform for the production of a wide range of recombinant proteins such as antibodies, enzymes, hormones, and vaccines

Identification and characterisation of coat proteins of plant COP vesicles

COP Vesikel sind am Proteintransport zwischen ER und Golgiapparat und innerhalb des Golgiapparates beteiligt. Sie besitzen auf ihrer Oberfläche eine Proteinhülle, die aus verschiedenen Proteinen aufgebaut wird. In der vorliegenden Arbeit wurden zwei Antikörper gegen Bestandteile der Proteinhülle pflanzlicher COPI und COPII Vesikel hergestellt. Mit diesen Antikörpern konnten gamma-COP und Sec23p als Bestandteile der pflanzlichen COP Vesikel charakterisiert werden. Außerdem wurden Immunlokalisierungen mit verschiedenen Antikörpern durchgeführt, die zeigten, dass COPI Vesikel und ihre Frachtmoleküle überwiegend im cis-Bereich des Golgiapparates pflanzlicher Zellen anzutreffen sind. Diese Vesikel weisen eine Größe von 50-60 nm auf und sind kleiner als COPI Vesikel aus Säugetierzellen

Exposure to TiO2 nanoparticles improves the physiological characteristics of drought‐challenged chickpeas (Cicer arietinum L.)

Abstract Drought stress markedly affects plant growth and crop production. In turn, treatment with some metal‐based nanoparticles (NPs) such as TiO2‐NPs could improve the plant tolerance against drought stress. In the present study, the effects of different levels of moisture regime (40%, 60%, and 90% field capacity [FC]) in conjunction with various concentrations of TiO2‐NPs (0, 5, 10, 20, and 40 mg. L−1) on chickpea were studied. Exposure of drought‐challenged chickpea plants to TiO2‐NPs raised antioxidant enzyme activity compared with plants grown under drought without TiO2‐NP treatment. The highest activity of ascorbate peroxidase (APX) was observed at 40% FC and application of 40 mg. L−1 TiO2‐NPs. Moreover, peroxidase (POX) activity has increased with the enhancing concentration of TiO2‐NPs to 20 mg. L−1 at 90% FC. In comparison, the application of 40 mg. L−1 TiO2‐NPs and decreasing levels of FC caused a rise in the activity of superoxide dismutase (SOD). Exposure to TiO2‐NPs raised the amount of total phenols and 2,2‐diphenyl‐1‐picryl‐hydrazyl‐hydrate (DPPH) at different levels of moisture regime. The content of malondialdehyde (MDA) at 60% FC has decreased by 22% after treatment with 20 mg. L−1 TiO2‐NPs compared with control plants. Also, treatment with TiO2‐NPs heightened the proline content, and the highest amount of proline was obtained at 40% FC by applying 20 mg. L−1 NPs. The treatment with TiO2‐NPs in the moisture regimes led to higher chlorophyll and carotenoid production in chickpea plants. Taken together, the application of TiO2‐NPs could raise the defense potential of chickpea plants against oxidative stress caused by the generation of reactive oxygen species

New Phenolics from Linum mucronatum subsp. orientale

Introduction: Lignans and flavonoids are widely distributed phenolics in the plant kingdom. Aryltetralin type lignans (podophyllotoxin derivatives) as the major secondary metabolites of Linum species play an important role in the production of chemotherapy drugs. In the present study, lignans and flavonoid glycosides from aerial parts of Linum mucronatum subsp. orientale were isolated and identified. Methods: The phytochemical investigation has been carried out on Hexane, DCM and MeOH extracts of the plant. Separation of chemical constituents was done using different chromatography (CC, prep-TLC, GC/MS and HPLC) methods. The major compounds of dichloromethane (DCM) and methanol extracts were isolated and their structures were elucidated using co-chromatography in the presence of known lignans, HPLC and NMR techniques. Results: Our results showed that podophyllotoxin and 6-MeO-α- peltatin, as new compound, are the major lignans of the DCM extract of L. mucronatum subsp. orientale. Two new flavonoid glycosides were also elucidated in the methanolic extract. Conclusion: The DCM and methanol extracts of L. mucronatum were found to contain aryltetralin-type lignans and flavonoids. The occurrence of 6-MeO-α- peltatin and flavonoids in L. mucronatum has been reported for the first time

Evaluating the Toxic Impacts of Cadmium Selenide Nanoparticles on the Aquatic Plant <em>Lemna minor</em>

Cadmium selenide nanoparticles (CdSe NPs) were synthesized by an easy and simple method and their properties were assessed by XRD, TEM and SEM techniques. The effects of CdSe NPs as well as Cd2+ ions on Lemna minor plants were investigated. The absorption of CdSe NPs by the plants had some adverse consequences that were assessed by a range of biological analyses. The results revealed that both CdSe NPs and the ionic form of cadmium noticeably caused toxicity in L. minor. Morphological parameters as well as peroxidase (POD) activity were deteriorated. In contrast, the activities of some other antioxidant enzymes (superoxide dismutase (SOD) and catalase (CAT)) as well as the contents of total phenol and flavonoids went up. Taken all together, it could be implied that CdSe NPs as well as Cd2+ were highly toxic to plants and stimulated the plant defense system in order to scavenge produced reactive oxygen species (ROS)