Computational design of fusion proteins against ErbB2-amplified tumors inspired by ricin toxin.

Although the anti-cancer activity of ricin is well-known, its non-specific targeting challenges the development of ricin-derived medicines. In the present study, novel potential ribosome-inactivating fusion proteins (RIPs) were computationally engineered by incorporation of an ErbB2-dependant penetrating peptide (KCCYSL, MARAKE, WYSWLL, MARSGL, MSRTMS, and WYAWML), a linker (either EAAAK or GGGGS) and chain A of ricin which is responsible for the ribosome inactivation. Molecular dynamics simulations assisted in making sure that the least change is made in conformation and dynamic behavior of ricin chain A in selected chimeric protein (CP). Moreover, the potential affinity of the selected CPs against the ligand-uptaking ErbB2 domain was explored by molecular docking. The results showed that two CPs (CP2 and 10) could bind the receptor with the greatest affinity.

Identification of Long Non-coding RNA Transcripts in Glycyrrhiza uralensis.

Background: Chinese liquorice (Glycyrrhiza uralensis), an important medicinal plant, contains various valuable secondary metabolites. Secondary metabolites biosynthesis is very tightly regulated; therefore, elucidation and manipulation of the biosynthetic pathways are of great interest. Recent studies have shown that lncRNAs play important regulatory roles in many biological processes, thus identification and modification of their expression is essential to metabolic pathways for biosynthesis of secondary metabolites. Objectives: In this study we attempted to identify non-coding RNA transcripts (lncRNAs) that may act as important regulators of diverse biological processes, including stress responses and developmental programs in Glycyrrhiza uralensis. Materials and Methods: Identification of potential lncRNAs in Chinese liquorice was performed using a bioinformatics pipeline from the available EST dataset of G. uralensis. Results: Bioinformatics analysis revealed that 1365 identical sequences in the range of 200 to 1286 base pair are putative lncRNAs. Only less than one percent of the predicted lncRNAs display sequence conservation with lncRNAs from other species. Moreover, 13 lncRNAs were detected as the potential precursors of 16 miRNAs. From this analysis, we also detected possible target genes of 16 known miRNA genes. The majority of the predicted miRNA target genes have important role in response to plant disease and a couple of them contribute to signalling and metabolic pathways. Conclusion: This study demonstrates the existence of lncRNAs in G. uralensis which has not been found before and provides valuable resources for further understanding and characterizing of lncRNAs and also a basis for additional investigation to reveal specific roles of lncRNAs in various biological processes and particularly in response to plant diseases.

In vitro regeneration and Agrobacterium-mediated genetic transformation of Dragon's Head plant (Lallemantia iberica).

Dragon's head plant (Lallemantia iberica), is a flowering species belongs to the mint family (Lamiaceae). The species contains valuable essential oils, mucilage and oil which are used in pharmaceutical and food industries. Tissue culture is a feasible strategy to attain large-scale production of plantlets with a huge potential to produce plants with superior quality. The objective of this study was to develop a simple and efficient method for regeneration and transformation of L. iberica. To reach this goal, the regeneration ability of various explants including leaf, cotyledonary node, hypocotyl and cotyledon segments was investigated in MS medium supplemented with diverse concentrations of NAA (Naphthalene acetic acid) and BAP (6-Benzyl Amino Purine). According to the results, cotyledonary nodes showed the best regeneration response. The maximum rate of regeneration (and number of induced shoots was achieved in 1 mg l-1 BAP in combination with 0.05 mg l-1 NAA from the cotyledonary nodes. Additionally, through the optimized regeneration technique Agrobacterium-mediated transformation of L. iberica was successfully accomplished. Gene transfer was assessed on leaf samples from regenerated plantlets under a fluorescent microscope to detect the GFP signals. Moreover, transgene integration and its expression were confirmed by PCR and RT-PCR analysis, respectively. The establishment of these efficient regeneration and genetic transformation methods paved the way for further application such as plant improvement, functional analysis and gene editing.

The spatio-temporal expression of some genes involved in the biosynthetic pathways of terpenes/phenylpropanoids in yarrow (Achillea millefolium).

Yarrow (Achillea millefolium) is a medicinal plant from the Asteracea which biosynthesize different secondary metabolites especially terpenes and phenylpropanoids. To improve our understanding of the regulatory mechanisms behind the biosynthesis of these compounds we analyzed the expression of some genes associated with the biosynthesis of terpenes and phenylpropanoids in different tissues and in response to trans-cinnamic acid (tCA) as an inhibitor of PAL activity. Isolation and expression analysis of DXR, GPPS, PAL and CHS genes together with linalool synthase (LIS) as monoterpene synthase was conducted in different developmental stages of leaves, flowers and in response to trans-cinnamic acid (tCA). Differential expression of these genes observed in different tissues. tCA up-regulated the biosynthetic genes of monterpenes and down-regulated the biosynthetic genes of phenylpropanoids. Gene expression analysis in intact leaves and leaves without glandular trichomes showed that DXR, LIS, PAL and CHS are highly expressed in glandular trichomes while GPPS expressed ubiquitously. Analysis of essential oils composition showed that sesquiterpenes and monoterpenes are main compounds; in which from 57 identified compounds the highest were germacreneD (% 11.5), guaiol (%10.38), spatulenol (%8.73) and caryophyllene oxide (%7.48).

Differential expression of 3ß-HSD and mlncRNAs in response to abiotic stresses in Digitalis nervosa.

Digitalis nervosa is an important medicinal plant species belonging to the family of Scrophulariaceae that has the potential to be used for heart failure. 3ß-hydroxysteroid dehydrogenase (3ß-HSD) is a key gene in the biosynthesis of cardenolides for making digitalis effective compounds, hence identification of this gene is important for genetic engineering purposes towards increasing the yield of cardiac glycosides. In addition, mRNA-like non-coding RNAs (mlncRNAs), a class of long non coding RNAs, play key roles in various biological processes and may affect cardenolides pathway in digitalis plants.  In the present work, full sequence of 3ß-HSD was isolated from Digitalis nervosa. Gene expression patterns of 3ß-HSD along with three mlncRNAs including mlncRNA23, mlncRNA28 and mlncRNA30 were studied and the results indicated that they are differentially expressed in different tissues including roots, stems and leaves, with the highest expression levels in leaves.  Moreover, the transcript levels of these genes affected by the cold and drought stresses. The results obtained from the present study is important in order to understand the potential role of mlncRNAs in digitalis plants, especially in response to abiotic stresses.

Inulin chain length modification using a transgenic approach opening new perspectives for chicory.

Chicory capable of synthesizing long-chain inulin is of great interest. During the growing season, the sucrose-sucrose 1-fructosyltransferase (1-SST) activity is vital for production of long-chain inulin in chicory. With the purpose to increase inulin chain length, we employed Agrobacterium-mediated transformation method. Transgenic chicory plants (Cichorium intybus L. var. sativum) cv. 'Melci' has been developed to overexpress sucrose-sucrose 1-fructosyltransferase (1-SST) under the control of the CaMV 35S promoter. The integration of the T-DNA into the plant genome was confirmed by PCR on genomic DNA using gene-specific primers. Quantification of the 1-SST transcript expression level revealed that transgenic plants showed higher 1-SST expression than those in non-transgenic plants. Further analyses proved that the fructan content of the roots significantly increased in the transgenic plants. These results revealed that overexpression of the 1-SST, the key gene in inulin biosynthesis in chicory, might serve as a novel approach to develop plants with the long-chain inulin content.

Tissue-specific gene-expression patterns of genes associated with thymol/carvacrol biosynthesis in thyme (Thymus vulgaris L.) and their differential changes upon treatment with abiotic elicitors.

Thyme (Thymus vulgaris L.) is known to produce a variety of phenolic monoterpenes such as thymol and carvacrol. Thymol and carvacrol are health-promoting, biocide and antitoxin compounds and have been considered as the main constituents of essential oils in T. vulgaris. To improve our understanding of the regulation of monoterpene biosynthesis in thyme, the expression of genes related to thymol and carvacrol biosynthesis in different tissues and in response to abiotic elicitors was analyzed. Methyl jasmonate (MeJA), salicylic acid (SA), trans-cinnamic acid (tCA) and UV-C irradiation were applied to T. vulgare leaves and transcript levels of early (DXR) and late (TvTPS1, CYP71D178 and CYP71D180) biosynthetic genes of thymol and carvacrol were measured. The results showed that early step and late step genes in thymol/carvacrol biosynthesis are differentially regulated. DXR was not found to be exclusively expressed in glandular trichomes; in contrast, biosynthetic genes including γ-terpinene synthase (TvTPS1) and two cytochrome P450s, CYP71D178 and CYP71D180, were preferentially expressed in glandular secretory trichomes. The high expression of late biosynthetic genes in glandular trichomes, which also contain the highest concentration of thymol and carvacrol, suggests that glandular trichomes are the structure in which thymol/carvacrol biosynthesis and accumulation occur. Our results indicate that in addition to abiotic elicitors, developmental and spatial factors also play a key role in the biosynthesis of thymol and carvacrol, most likely relating to glandular trichome density and/or activity. Hence optimization of these factors could be considered as a useful strategy to achieve high yield of valuable compounds in T. vulgare or other closely related plant species.

Parthenolide accumulation and expression of genes related to parthenolide biosynthesis affected by exogenous application of methyl jasmonate and salicylic acid in Tanacetum parthenium.

KEY MESSAGE: Up-regulation of germacrene A synthase and down-regulation of parthenolide hydroxylase genes play key role in parthenolide accumulation of feverfew plants treated with methyl jasmonate and salicylic acid. Parthenolide is an important sesquiterpene lactone due to its anti-migraine and anti-cancer properties. Parthenolide amount was quantified by high-performance liquid chromatography after foliar application of methyl jasmonate (100 µM) or salicylic acid (1.0 mM) on feverfew leaves in time course experiment (3-96 h). Results indicate that exogenous application of methyl jasmonate or salicylic acid activated parthenolide biosynthesis. Parthenolide content reached its highest amount at 24 h after methyl jasmonate or salicylic acid treatments, which were 3.1- and 1.96-fold higher than control plants, respectively. Parthenolide transiently increased due to methyl jasmonate or salicylic acid treatments until 24 h, but did not show significant difference compared with control plants at 48 and 96 h time points in both treatments. Also, the transcript levels of early pathway (upstream) genes of terpene biosynthesis including 3-hydroxy-3-methylglutaryl-coenzyme A reductase, 1-deoxy-D-xylulose-5-phosphate reductoisomerase and hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase and the biosynthetic genes of parthenolide including germacrene A synthase, germacrene A oxidase, costunolide synthase and parthenolide synthase were increased by methyl jasmonate and salicylic acid treatments, but with different intensity. The transcriptional levels of these genes were higher in methyl jasmonate-treated plants than salicylic acid-treated plants. Parthenolide content measurements along with expression pattern analysis of the aforementioned genes and parthenolide hydroxylase as side branch gene of parthenolide suggest that the expression patterns of early pathway genes were not directly consistent with parthenolide accumulation pattern; hence, parthenolide accumulation is probably further modulated by the expression of its biosynthetic genes, especially germacrene A synthase and also its side branch gene, parthenolide hydroxylase.

Validation of reference genes for gene expression analysis in chicory (Cichorium intybus) using quantitative real-time PCR.

BACKGROUND: Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) is a sensitive technique for quantifying gene expression levels. One or more appropriate reference genes must be selected to accurately compare mRNA transcripts across different samples and tissues. Thus far, only actin-2 has been used as a reference gene for qRT-PCR in chicory, and a full comparison of several candidate reference genes in chicory has not yet been reported. RESULTS: Seven candidate reference genes, including nicotinamide adenine dinucleotide dehydrogenase (NADHD), actin (ACT), beta-tubulin (TUB), glyceraldehyde-3-phosphate-dehydrogenase (GADPH), histone H3 (H3), elongation factor 1-alpha (EF) and 18S rRNA (rRNA) were selected to study the expression stability for normalisation of gene expression in chicory. Primer specificity and amplification efficiency were verified for each gene. The expression stability of these genes was analysed across chicory root and leaf tissues using geNorm, NormFinder and BestKeeper software. ACT, EF, and rRNA were the most stable genes as identified by the three different analysis methods. In addition, the use of ACT, EF and GAPDH as reference genes was illustrated by analysing 1-FEHII (FEHII) expression in chicory root and leaf tissues. These analyses revealed the biological variation in FEHII transcript expression among the tissues studied, and between individual plants. CONCLUSIONS: geNorm, NormFinder, and BestKeeper analyses indicated that ACT, EF and rRNA had the highest expression stability across leaf and root tissues, while GAPDH and NADHD showed relatively low expression stability. The results of this study emphasise the importance of validating reference genes for qRT-PCR analysis in chicory. The use of the most stable reference genes such as ACT and EF allows accurate normalisation of gene expression in chicory leaf and root tissues.