The potential of gamma irradiation on antioxidant capacity and genomic alterations in Calendula officinalis.

There are lines of evidence that ionizing radiations such as gamma rays can cause different biological effects on plants. Marigold (Calendula officinalis L.) is a member of the family Asteraceae. It possesses profound amounts of active ingredients. The aim of this study was to evaluate the changes imposed upon different dose levels of gamma radiation on some features of Calendula officinalis such as antioxidant activity, total phenolic compounds and flavonoid contents, antibacterial activity and genomic alterations. Calendula officinalis seeds were exposed to different doses of Gamma radiation (0, 10, 15, 20 and 25 GY). Total phenolics, flavonoids, antioxidant activity (measured by DPPH assay) using methanolic extracts of plants and antibacterial activity measured by the disc diffusion assay showed significant differences to the control samples. The samples treated with 10 GY gamma rays showed the highest total phenol and flavonoid contents. Antioxidant activity significantly differed between Gamma rays dose levels and it was the highest at 25 GY. Four bacterial strains including E. coli, Bacillus subtilis and Pseudomonas aeroginosa were used for the antibacterial assay. Extracts from plants treated with 25 GY gamma rays showed the highest antibacterial activity against the 4 bacterial strains. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to study the genetic variation. The polymorphism information content (PIC) for RAPD primers ranged from 3% to 13% and ranged from 6 to 13% for ISSR primers. Results indicated that ISSR markers were more efficient than RAPD markers, as they detected 25.57% polymorphic DNA bands compared to 21.31% polymorphism for RAPD markers.

Identification of drought-tolerant hub genes in Iranian KC-2226 genotype of Aegilops tauschii using transcriptomic analysis.

Aegilops tauschii, as a donor of D genome to the bread wheat with a valuable source of resistance to different biotic and abiotic stresses, is used to improve the quality of wheat cultivars. Every genotype has a specific genetic content, the investigation of which can lead to the identification of useful genes such as stress tolerance genes, including drought. Therefore, 23 genotypes of Ae. tauschii were selected to evaluate their morphological and physiological traits under greenhouse conditions. Among them, a superior tolerant genotype (KC-2226) was chosen for transcriptomic analysis. Our result showed that 5007 and 3489 genes were deferentially up- and downregulated, respectively. Upregulated genes were involved in photosynthesis, glycolysis/gluconeogenesis, and amino acid biosynthesis whereas downregulated genes were often engaged in DNA synthesis, replication, repair and topological changes. The result of protein-protein interaction network analysis showed that AT1G76550 (1.46), AT1G20950 (1.42), IAR4 (1.19), and PYD2 (1.16) among upregulated genes and THY-1 (44), PCNA1 (41) and TOPII (22) among down-regulated genes had the highest interactions with other genes. In conclusion, Ae. tauschii employs elevated transcription of specific genes involved in photosynthesis, glycolysis and gluconeogenesis and amino acid biosynthesis pathways rather than genes active in DNA synthesis and repair to provide the energy needed for the plant to survive under stress conditions.

Effect of iron and silver nanoparticles on coenzyme Q10 production by Gluconobacter japonicus FM10.

BACKGROUND AND OBJECTIVES: Coenzyme Q10 is an anti-aging agent whose demand is increasing progressively. There are various strategies used for increasing coenzyme Q10 production by microorganisms. In this study, for the first time, we investigated the effect of iron oxide and silver nanoparticles on coenzyme Q10 production by Gluconobacter japonicus FM10. MATERIALS AND METHODS: In the first step, a preliminary experiment was set and carried out to obtain the minimum inhibitory concentrations of the nanoparticles on the strain FM10. Then the sub-MIC concentrations were used to investigate their effect on coenzyme Q10 production in the stationary and exponential phases of the growth, separately. RESULTS: The results showed that coenzyme Q10 production increased in the presence of the iron oxide and silver nanoparticles. The silver nanoparticles induced 1.9 times higher coenzyme Q10 production. The highest level of coenzyme Q10 was induced when the silver nanoparticles were added to the culture medium at the stationary phase. CONCLUSION: This should be noticed that so far nanoparticles have been considered as antibacterial agents, rather than being considered to cause probable beneficial effects on the induction of useful products in the microbial world. In this regard, their potential for increasing coenzyme Q10 production has received no attention. However, our present results showed that the nanoparticles can be used to increase the production efficiency of coenzyme Q10 in Gluconobacter.

Introducing a thermotolerant Gluconobacter japonicus strain, potentially useful for coenzyme Q10 production.

In this report, Gluconobacter strains were screened for coenzyme Q10 (CoQ10) production. A thermotolerant strain, Gluconobacter japonicus FM10, was eventually employed for CoQ10 production optimization. To do so, a two-step optimization strategy was used. The first step focused on biomass increase and the second step focused on increase in CoQ10 production. Factors including temperature, pH, carbon, and nitrogen sources were optimized at the first step, and temperature, pH, and aeration were optimized at the second step. The batch culture fermentation was used with the optimized factors of the first phase (30 °C, pH 6.5, D-sorbitol, and yeast extract-peptone as the carbon and nitrogen sources). After 18 h, the temperature, pH, and aeration were shifted to the optimized values of the second step (36 °C, pH 7, and no aeration). By this strategy, the dry cell mass (17.1 g/L) and CoQ10 (23.2 mg/L) were obtained after 20 h, which the latter was 2.3 times higher than that of the first step of optimization. Among the conditions tested, carbon source was the most important factor on the cell growth at the first step while no aeration was the key factor for CoQ10 production in the second step of optimization.

Constructing and transient expression of a gene cassette containing edible vaccine elements and shigellosis, anthrax and cholera recombinant antigens in tomato.

Shigella dysenteriae causing shigellosis is one of the diseases that threaten the health of human society in the developing countries. In Shigella, IpaD gene is one of the key pathogenic genes causing strong mucosal immune system reactions. Anthrax disease is caused by Bacillus anthracis. PA protective antigen is one of the subunits in anthrax toxin complex responsible for the transfer of other subunits into the cytosol of host cells. The 20 kDa subunit of PA (PA20) has the property of immunogenicity. CTxB or B subunit of Vibrio cholerae toxin (CT) is a non-toxic protein and has the function to transfer toxic subunit into cytosol of the host cells by binding to GM1 receptor. The aim of this study was to fuse PA20, ipaD and CTxB and transform tomato plants by this cassette in order to produce an oral vaccine against shigellosis, anthrax and cholera. CTxB was used for these two antigens as an immune adjuvant. IpaD and PA20 genes were cloned in pBI121 containing the CTxB gene and Extensin signal peptide. In order to evaluate the transient expression of Shigellosis, Anthrax and Cholera antigens, agro-infiltrated tomato tissues were inoculated with Agrobacterium tumefaciens containing the gene cassette. Cloning was confirmed by PCR, enzymatic digestion and sequencing techniques. Expression of the antigens was examined by SDS-PAGE, dot blot and ELISA. Maturate green fruits demonstrated the highest expression of the recombinant proteins. The first phase of this study was carried out for cloning and expressing of CtxB, ipaD and PA20 antigens in tomato. In the next phase, we aim to analyze the immunogenicity of this vaccine candidate in laboratory animals.

A quick, efficient, and cost-effective method for isolating high-quality total RNA from tomato fruits, suitable for molecular biology studies.

Tomato (Solanum lycopersicum L.) is the primary model for the study of fleshy fruits, and research on this species has elucidated many aspects of fruit physiology, development, and metabolism. However, for advancing such studies at molecular biology levels, the RNA isolation from fruit tissues is often essential. The RNA isolation from tomato fruits is complicated because of the presence of high levels of polysaccharides, polyphenolics, pigments, and secondary metabolites and also the varying water content during development. Here, we present an optimized protocol for the isolation of total RNA from the fruit tissues at different developmental stages. In comparison to the previous methods described for the RNA isolation from tomato fruit, this method has the advantages that it does not involve the use of guanidine salts, lyophilizers, and commercial reagents, reduces the time and cost of extraction, overcomes the high water content problem, and promotes RNA quality by inhibiting RNA degradation and minimizing the gDNA, polyphenolic and polysaccharide contaminations. Using this method, high yields of high-purity and intact RNA samples were obtained as confirmed by the spectrophotometric readings and the electrophoresis on denaturing agarose gels. The isolated RNA was employed as a robust template for cDNA synthesis, reverse transcriptase-polymerase chain reaction (RT-PCR), and temporal gene expression analysis. The functionality of the isolated RNA was further demonstrated through cloning full-length cDNAs encoding ß-galactosidase proteins by RT-PCR and sequencing.

Identification of Chlorophyceae based on 18S rDNA sequences from Persian Gulf.

BACKGROUND AND OBJECTIVES: Chlorophyceae are important constituents of marine phytoplankton. The taxonomy of Chlorophyceae was traditionally based solely on morphological characteristics. In the present research project, genetic diversity was investigated to analyze five species of Chlorophyceae from waters of the Persian Gulf. MATERIALS AND METHODS: A clone library of the ribosomal small subunit RNA gene (18S rDNA) in the nuclear genome was constructed by PCR, and then, after examining the clones, selected clones were sequenced. The determined clone sequences were analyzed by a similarity search of the NCBI GenBank database using BLAST. RESULTS AND CONCLUSION: Eleven sequences were identified correctly and used for phylogenetic analysis. We identified species of Chlorophyta (Chlorella sorokiniana, Chlamydomonas sp., Neochloris aquatic, Picochlorum sp. and Nannochloris atomus) without the need to conduct extensive colony isolation techniques. Therefore, this improved molecular method can be used to generate a robust database describing the species diversity of environmental samples.

The effect of gamma irradiation on astaxanthin synthetase encoding gene in two mutant strains of Phaffia rhodozyma.

BACKGROUND AND OBJECTIVES: Astaxanthin, an orange-red carotenoid pigment, acts as a protective agent against oxidative damage to cells in vivo. The astaxanthin synthetase gene (crtS) size consists of 3995 bp. This gene has been suggested to catalyse ß-carotene to astaxanthin in Phaffia rhodozyma. The aim of this research was to find any possible changes in this gene in two mutant strains, Gam1 and Gam2 (with high astaxanthin pigment production), previously created by gamma irradiation. MATERIALS AND METHODS: The astaxanthin synthetase gene sequence of Phaffia rhodozyma in the NCBI Gene bank was used to design primer. In Gam1, this gene was amplified using primers Asta F1, Asta R2, Asta F3, Asta R4. In Gam2, primers asta F1, asta R4 were used to amplify the gene. The amplified fragments were 8 sequenced using primers Asta F1, Asta R1, Asta F2, Asta R2, Asta F3, Asta R3 and Asta F4, Asta R4. Astaxanthin synthetase gene from two mutant strains, Gam1 and Gam2 were amplified using PCR. The amplified products were sequenced and aligned using the ClustalW software. CONCLUSION: The comparison of this gene showed 98% and 99% similarities between the reference sequence and Gam1 and Gam2 mutant strains, respectively, whereas the comparison of this gene in Gam1 and Gam2 mutant strains showed 97% similarity. However, the deduced proteins showed 78% and 83% between the reference protein obtained from the wild type and Gam1 and Gam2, respectively. This similarity was 75% between the mutant strains.

Cloning, identification and expression analysis of ACC oxidase gene involved in ethylene production pathway.

1-aminocyclopropane-1-carboxylic acid oxidase (ACO) enzyme is a member of the Fe II-dependent family of oxidases/oxygenases which require Fe(2+) as a cofactor, ascorbate as a cosubstrate and CO(2) as an activator. This enzyme catalyses the terminal step in the plant signaling of ethylene biosynthetic pathway. A 948 bp fragment of the ACO1 gene cDNA sequence was cloned from tomato (Lycopersicon esculentum) fruit tissues by using reverse transcriptase-polymerase chain reaction (RT-PCR) with two PCR primers designed according to the sequence of a tomato cDNA clone (X58273). The BLAST search showed a high level of similarity (77-98 %) between ACO1 and ACO genes of other plants. The calculated molecular mass and predicted isoelectric point of LeACO1 were 35.8 kDa and 5.13, respectively. The three-dimensional structure studies illustrated that the LeACO1 protein folds into a compact jelly-roll motif comprised of 8 α-helices, 12 ß-strands and several long loops. The cosubstrate was located in a cofactor-binding pocket referred to as a 2-His-1-carboxylate facial triad. Semi-quantitative RT-PCR analysis of gene expression revealed that the LeACO1 was expressed in fruit tissues at different ripening stages.

Gamma irradiation as a useful tool for the isolation of astaxanthin-overproducing mutant strains of Phaffia rhodozyma.

Astaxanthin is a carotenoid pigment responsible for the red color of the flesh of many marine animals. There is an increasing interest in the use of astaxanthin in aquaculture, chemical, pharmaceutical, and alimentary industries. Phaffia rhodozyma has been identified as the best biological source of astaxanthin. Mutagenesis was carried out using different doses of gamma irradiation (1.0, 2.0, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, and 7.0 kGy), and 10 mutant colonies (Gam1-Gam10) were obtained. Highly pigmented mutant strains produced astaxanthin at approximately 15 887.5 µg/L dry mass of yeast, whereas the parental strain produced it at 1061.64 µg/g dry mass of yeast. In the thin-layer chromatography analysis, P. rhodozyma JH-82 and Gam1 mutant strain produced the same retention factor (R(f)) values, but Gam1 showed a higher astaxanthin content than JH-82.

Impact of gamma rays on the Phaffia rhodozyma genome revealed by RAPD-PCR.

BACKGROUND AND OBJECTIVES: Phaffia rhodozyma is a red yeast which produces astaxanthin as the major carotenoid pigment. Astaxanthin is thought to reduce the incidence of cancer and degenerative diseases in man. It also enhances the immune response and acts as a free-radical quencher, a precursor of vitamin A, or a pigment involved in the visual attraction of animals as mating partners. The impact of gamma irradiation was studied on the Phaffia rhodozyma genome. MATERIALS AND METHODS: Ten mutant strains, designated Gam1-Gam10, were obtained using gamma irradiation. Ten decamer random amplified polymorphic DNA (RAPD) primers were employed to assess genetic changes. RESULTS: Nine primers revealed scorable polymorphisms and a total of 95 band positions were scored; amongst which 38 bands (37.5%) were polymorphic. Primer F with 3 bands and primer J20 with 13 bands produced the lowest and the highest number of bands, respectively. Primer A16 produced the highest number of polymorphic bands (70% polymorphism) and primer F showed the lowest number of polymorphic bands (0% polymorphism). Genetic distances were calculated using Jaccard's coefficient and the UPGMA method. A dendrogram was created using SPSS (version 11.5) and the strains were clustered into four groups. CONCLUSION: RAPD markers could distinguish between the parental and the mutant strains of P. rhodozyma. RAPD technique showed that some changes had occurred in the genome of the mutated strains. This technique demonstrated the capability to differentiate between the parental and the mutant strains.