Iranian Journal of Biotechnology
Volume:19 Issue: 4, Autumn 2021

Sclerotinia sclerotiorum (Ss) is a broad host range necrotrophic ascomycete fungus affecting over 400 plant species. Ss causes stem rot disease on Camelina sativa (Cs) an allohexaploid crucifer species that is promoted as a low input crop and industrial oil attributes suitable as biofuel and lubricant. Histochemical and molecular studies has linked resistance to Ss in C. sativa with the cell wall lignification (Eynck et al., 2012) and reported constitutive expression of Cinnamoyl-CoA Reductase 4 (CsCCR4) gene, in the Cs resistant line CN114263. Modern breeding efforts, such as gene editing, are needed to improve commercial lines and to limit the risk of crop loss which would be substantial to producers. To investigate the importance of monolignol biosynthesis and the role of CsCCR4 in Camelina resistance to Ss we generated CsCCR4 knockout mutants of CN114263 Camelina line using CRISPR/Cas9-mediated gene editing. Thirty T1 plants were produced via floral dip transformation followed by glyphosate spraying that was used in the first step of screening procedures and were confirmed by PCR method. Transgene’s T-DNA copy number variation, T-DNA CNV, in T1 and T2 progenitors were determined using digital droplet PCR (ddPCR) and the occurrence of mutation in the three copies of CsCCR4 homeologues in T1 and T2 generations were scrutinized by drop-off assay technique. To make sure that if the created mutants in T2 plants are real, TOPO TA sequencing flanking the Cas9/gRNA specific hot point of cleavage for three of them was conducted. In the T1 generation, 25 plants were confirmed which had between one to nine T-DNA copies in the corresponding Camelina genome. In T2 generation the population were screened for potential mutation in CsCCR4 gene. Various types of mutations, including insertions and deletions, were demonstrated in three copies of CsCCR4. In fact, CRISPR system could have cut one, two or three copies of the gene in events numbered T2-plant 10, T2-plant 15 and T2-plant 19, respectively. The T3-plant 19 which showed mutation in all versions of CsCCR4 in previous generation had susceptibility to S. sclerotiorum invasion and was kept as real CsCCR4 mutant material for further investigations of Camelina-Sclerotinia interaction. Mutation in CsCCR4 had occurred through error-prone none- homologous end joining (NHEJ) nucleus DNA repair pathway. Ss challenge on the early flowering T3 generation. The T3 plants with mutation causing premature stop codon at position 217 of CsCCR4 were compromised in their resistance to Ss compared to the wildtype resistant control parent CN114263. Using ddPCR it easily was possible to identify both the T-DNA CNV and occurrence of mutation in CsCCR4 homeologues in T1 and T2 progenitors. We illustrated that CRISPR/Cas9-mediated mutation is a decent technique that can be utilized to expedite the mutant line development which could assist to figure out the activity of a CsCCR4 gene in defense responses to the pathogens in C. sativa as prospective oilseed crop for biodiesel production.

Insulin-like growth factor-1 (IGF-1), in addition to having insulin-like effects, has boosting effects on all cells in human body. Most of the recombinant IGF-1 required for patients suffering from its deficiencyis currently produced by bacterial and yeast systems. Plant systems, especially chloroplasts, have many benefitsfor producing human blood proteins. Production costs are low in these systems, and their side effects are less than other systems. In this study, the transfer and expression of mature IGF-1 protein cDNA in tobacco chloroplasts under the control of strong plastid transcription and translation elements was evaluated. The biolistic transformation method was used to transfer the IGF-1 gene cloned into the pRB94-IGF1 chloroplast vector (1). Homoplasmic transplastomic plants were produced through four selection rounds on the selective medium. Transfer of foreign genes to chloroplast genome was confirmedby PCR, Southern blotting and seed progeny test. RT-PCR and SDS-PAGE methods were used to evaluate the expression of IGF-1 gene in transgenic line. A truly transformed line was identifiedfrom selected seedlings by PCR method. The seed progeny test of 4th-regeneration-round transgenic plants of this line showed maternal inheritance and homoplasmic level for the selectable marker gene, which confirmsthe transfer and expression of the marker gene in the chloroplast genome. The Southern blot test also confirmedthe transfer of the IGF-1 gene into the chloroplast genome. RT-PCR test showed that IGF-1 gene transcription is performed correctly in transgenic plants. Finally, accumulation of IGF-1 protein in transgenic plants was detected by SDS-PAGE. Successful transfer and expression of the native human IGF-1 gene in tobacco chloroplast genome is reported.

Since embryogenesis, plants deal with environmental changes, which might affect their growth and development. Plant autophagy has been shown to function in various stress responses, immunity, development, and senescence. Acquired thermotolerance or thermopriming is enhanced resistance to the elevated temperature following heat stress. Potential contribution of autophagy mechanism after thermopriming was investigated in shoot apical meristem (SAM) of Arabidopsis thaliana. Transcriptic expression of Autophagy related Genes (ATGs) were analyzed by qRT-PCR data in 5-day old Arabidopsis thaliana (Col0) seedlings at 4 h and 24 h after thermopriming. Autophagy induction was confirmed by confocal microscopy. Expression patterns of 39 ATGs and ATG-receptors were described and relevant thermopriming induced autophagy genes were identified according to their highest expression fold changes during the time after treatment. Significantly, ATG8A, ATG8B, ATG8G, ATG8H, ATI1, ATI2, NBR1, and TSPO genes were identified as the most relevant thermopriming-associated autophagy genes especially in SAM of young seedlings. This mainly implies the role of ATG8 core proteins and their receptor interactors in the regulation of autophagy in form of selective or non-selective during environmental stresses. Autophagy, a conserved mechanism for cell survival in plants will be activated in response to the thermopriming which is a promoted acquired resistance stimulus. Determined key genes and components of autophagy associated with thermal priming signaling pathway could be noteworthily employed to study transcriptional regulation of autophagy and integrated defense system against environmental stresses for the improvement of plant thermal tolerance and resistance to the pathogens.

Chitosan (CS) nanoparticles have attracted considerable attention as a non-viral and cationic carrier for delivery of therapeutic proteins and antigens and offer non-invasive routes of administration such as oral, nasal and ocular routes, and also show adjuvant characteristics for vaccines. Preparation and formulation of CS nanoparticles as a capable carrier with immunoadjuvant properties to enhance the bioavailability of antigen and produce antibody with high affinity. CS nanoparticles were produced by ionic gelation process of sodium tripolyphosphate (TPP) with CS. Particle size and morphology of nanoparticles were determined using Dynamic Light Scattering (DLS) and Scanning Electron Microscopy (SEM) and also direct observation under light microscope. The influence of the initial BSA concentration and CS concentration on loading efficiency and release behavior was evaluated. The ε-toxin (derived from Clostridium perfringens type D) was loaded on CS nanoparticles and the complex was injected hypodermically into the rabbits for once. The anti ε-toxin antibody level in blood serum was evaluated using Dot Blot and ELISA methods. The CS nanoparticles in different groups have a particle diameter (Z-average) in approximate ranges of 200-400, 300-600, 450-800 nm and a positive Zeta potential (32.4 - 48.6 mv). Optimum loading efficiency was achieved for CS at a concentration of 0.5 mg.mL-1 and TPP of 1.0 mg.mL-1. The results showed that the toxin-CS complex produces antitoxin at levels more than twice as high the control. The CS nanoparticles can be used as a good biodegradable carrier for protein and antigen delivery.

Increased economic competitiveness in the biopharmaceutical industry requires continuous improvement of bioprocesses. In this regard compositions of fermentation media have an important role in bioprocesses.

The modification of the culture medium has proven effective in enhancing the yield and productivity of fermentation processes. The objective was to investigate the influence of castor oil as the main carbon source for Saccharopolyspora erythraea, on the yield of antibiotic fermentative production.

The titer of erythromycin was evaluated in Saccharopolyspora erythraea cultures, containing various concentrations of castor oil, in comparison to the control culture containing rapeseed oil.

The results showed an enhancement in erythromycin production when 50 g.L-1and 40 g.L-1of castor oil were added to the fermentation culture instead of rapeseed oil, respectively. The highest amount of production was obtained on the eleventh day of fermentation time in all media.

Erythromycin production in the control medium was relatively less than that of the treatments, indicating that S. erythraea consumed castor oil as a rich alternative carbon source. The results show that castor oil was more suitable as a carbon source for erythromycin production than a medium containing rapeseed oil.

MicroRNA (miRNA) is a key regulator at the gene posttranscriptional regulation level. We have previously identified miRNAs and their putative targets in 3 representative red algae, Chondrus crispus, Galdieria sulphurariais and Porphyridium purpureum. In this study, unique molecular and evolutionary characterization of miRNAs were revealed in the 3 red algae based on the comparative miRNAs profiling. Genome locations of small RNAs (sRNAs), miRNAs and MIRNAs (MIRs) in the 3 red algae were shown by collinearity analysis. Characterization of miRNAs and MIRs were profiled via bioinformatics analysis. Taken MIR156s and miR156s for examples, red algae miRNAs evolutionary features were demonstrated via phylogenetic and evolutionary information analysis. MiRNA targets main inhibition type was validated via performing data statistics and RLM-RACE PCR. Key target genes and their function were predicted by the common Gene Ontolgoy (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Quantity, nucleotide bias and common sequences of miRNAs were analyzed in the 3 red algae. Four typical precursor structures and primary molecular features of red algae miRNAs were profiled. Genome-wide collinearity analysis of sRNAs, miRNAs and MIRs in the 3 red algae was performed to show their distribution and interrelation based on the deep sequencing data. Taken red algae MIR156s for example, their family members and sequences divergence were demonstrated. The whole evolutionary processes of miR156s and pre-miR156s in red algae were steady with negative selected pressure though diverse phylogenetic relationships and evolutionary parameters showed. Through 3 red algae miR156 targets validation, cleavage was validated as their main miRNA targets inhibition type. The common target genes (GO:0009536) enriched significantly for plastid formation will provide important insights for red algal biopigment research. The common KEGG pathways (ko01100) enriched significantly were predicted without a detailed reference metabolic map. MiRNA plays an essential role in gene expression regulation involved in diverse biological processes of red algae. Comprehensive molecular and evolutionary features of miRNAs in the 3 red algae will provide insights for further utilizing the algae resources at the molecular level.

Urea with super-hydrating and moisturizing properties is mainly used as an adjunctive treatment of diseases associated with dry skin. In this regard, the recombinant human growth hormone (rhGH) with rejuvenating properties is used as a base material in beauty creams. Although urea easily passes through the skin, the epidermal skin barrier restricts the passage of hGH due to its size. in this research, in order to solve this problem, hydroxy propyl-beta cyclodextrin (HP-β-CD) is used as a soluble chemical enhancer. UV and circular dichroism spectroscopy were used for the investigation of structural modification. The permeation process was studied in vitro on rat skin using vertical Franz diffusion cells. Enzyme-linked immunosorbent assay were used for rhGH activity assessment and evaluation of transdermal delivery. First, due to the denaturing effects of urea on proteins its concentration was optimized to maintain biological structure and protein activity. UV spectroscopy and CD data proved that the secondary structure of rhGH is preserved in the presence of urea (0.5-2 M) and HP-β-CD, which elevates urea and rhGH permeation. Maximum permeability was observed at 120 min after sampling (1424.35 ng.ml.cm-2), which was much higher than the control. Using a higher concentration of urea in the formulation will significantly decrease the level of rhGH delivery. According to results, this strategy can be considered as a successful method for enhanced Co-delivery of rhGH and urea.

Pyridoxal 5ʹ -phosphate synthase (PLPS) is present in deoxyxylose 5ʹ-phosphate-independent of the de novo vitamin B6 biosynthesis pathway. This enzyme complex consists of PdxS and PdxT, which function as synthase and glutamine amidotranferase respectively to produce PLP. This study aimed to clone, express, and purify PLPS of Geobacillus sp. H6a, followed by its characterization. The PdxS and PdxT genes were amplified from Geobacillus (Gh) sp. H6a. Recombinant vectors pET28a-GhpdxS and pET28a-GhpdxT were constructed and the resulting His-tagged proteins were expressed in E. coli BL21(DE3). The soluble rGhpdxS and rGhpdxT were purified via nickel-affinity chromatography and cation-exchange chromatography. The mixture of rGhpdxS and rGhpdxT was further characterized. The molecular weights of rGhpdxS and rGhpdxT were estimated to be 35 and 23 kDa by SDS-PAGE, respectively. The native form of rGhpdxS showed hexamer and dodecamer, whereas those of rGhpdxT were a monomer upon detection with non-denaturing gel electrophoresis and gel filtration. A molar ratio of 1:1 of rGhpdxS:rGhpdxT showed the highest PLP synthesis activity (4.16 U.mg-1) and was used for analyzing the biochemical properties. The kinetic values were obtained by using glyceraldehyde 3-phosphate, ribose 5-phosphate, and glutamine as the substrates. The rGhPLPS showed pentose phosphate isomerization without triose phosphate isomerase activity. The metal ions affected PLP synthesis activity. The optimum pH and optimum temperature of rGhPLPS were 9 and 70 °C, respectively. The rGhPLPS was active over a broad range of temperatures and pH values. These results support the potential of rGhPLPS as a candidate for industrial application.

Cancer is a major health concern in human populations worldwide, and due to its causes being multi-factorial, it is not easily curable. Many attempts have been made to tackle this disease in hopes of finding effective anticancer agents which are not harmful to healthy tissues. Peptides with several medicinal activities have been shown to be good candidates as anticancer agents to replace common classic anticancer drugs. Peptides in conjugation with either biologically active heterocyclic compounds or anticancer drugs may result in new molecules compiling the biological benefits of both individual compounds within a unit structure. In this study some triazole-peptide conjugates as well as ciprofloxacin-peptide conjugates were designed, synthesized, and their anticancer activities evaluated. A normal skin cell line, NIH3, was also employed to determine the safety profiles of these conjugates. Two peptides; YIGSR and LSGNK were synthesized by the solid phase peptide synthesis (SPPS) method using Wang resin. Cell viability was examined by employing the MTT assay. To determine the cytotoxicity of the triazole and ciprofloxacin conjugates, two human cancer cell lines were employed; HepG2 (human liver cancer cell line) and LNCaP (human prostatic carcinoma cell line). A human skin fibroblast cell line was also included for comparison. MTT results showed that all the compounds could inhibit the viability of cancerous cells in a concentration- dependent manner. The results showed that these peptide conjugates are toxic against the aforementioned cancerous cells and thus may raise a hope for finding new anticancer agents made by such strategy in the near future.