مجله بیوتکنولوژی کشاورزی
سال سیزدهم شماره 1 (پیاپی 42، بهار 1400)

Scrophularia striata is a plant belonging to the Scrophulariaceae family. Due to the importance of phenylpropanoid compounds in this plant, identification and isolation of genes encoding cinnamate enzymes 4-hydroxylase (C4H) and chalcone synthetase (CHS) involved in the biosynthesis of these compounds and also, their expression under the influence of various abiotic elicitors was performed.

Seeds of wild plants grown around the university were collected and were cultured in greenhouse conditions. Sampling was done before flowering stage. RNA extraction, cDNA synthesis, isolation and sequencing of CHS and C4H genes in the roots of this plant were successfully performed for the first time. The expression of these genes was performed under the influence of three salicylic acid (SA), jasmonic acid (JA) and gibberellic acid (GA) each at concentrations of 100 and 300 ppm using a completely randomized design at the transcript level by Real Time PCR method.

The results showed that among the elicitors used, JA and SA with the concentration of 100 ppm had the greatest effect on increasing the expression of C4H and CHS genes so that in investigating the effect of the above-mentioned elicitors on the accumulation of phenylpropanoid compounds, this issue was partially confirmed. Also, in the study, increasing the concentration of JA and SA decreased the expression of C4H and CHS genes.

The general results of the use of elicitors on gene expression and accumulation of secondary metabolites showed that the external use of stimulants in appropriate concentrations, possibly through the induction of the plant immune system, can play a role in the biosynthesis and accumulation of valuable secondary metabolites such as phenylpropanoid compounds in the roots of Scrophularia striata and subsequently identifying and analyzing the expression of genes involved in the biosynthesis of such valuable compounds can be effective in complementary basic research for use in the pharmaceutical and food industries.

Due to high cytotoxicity effect, ribonucleases (RNases) are known as pharmacological agents with therapeutic potential. The high potential of these enzymes in the destruction of RNA strand and their other substrates and subsequently, cell destruction or cessation of cell division causes a very high cytotoxic effect of this enzyme in various cancer cells. Hence, this review investigates the therapeutic applications of ribonucleases, cell pathways and their mode of action as cytotoxic agents in order to pay attention to them in researches related to the health and therapy fields.

In this study the keywords such as cancer, ribonuclease, ribonuclease inhibitor and RNA were used to search in databases including Scopus, SID, IranDoc, PubMed, Google Scholar, Web of Science and IranMedex. In order to select the documents used, all articles published in non-English and Persian languages, duplicate articles, articles that could not be accessed to the full text, as well as articles that were presented as abstracts were removed. Finally, the selected cases were thoroughly studied and summarized in order to prepare the current review.

The study and review of research conducted showed that ribonucleases have the ability to kill tumor cells and that these enzymes have antiviral properties. Bovine pancreatic ribonuclease (RNase A), bovine seminal ribonuclease (BS-RNase), Onconase, and angiogenin are known as RNases with high antitumor activity which exert cytotoxic activity on cancer cells selectively by involving different cellular pathways and/ or enhance the cytotoxicity by mutation. Also, the study of articles related to the function of ribonucleases showed that the investigation of genetic pathways of synthesis and mechanisms of cytotoxicity in these enzymes will provide the development of new pharmaceutical products in the future. Utilization of engineering processes and chemical changes in ligand / receptor structure may increase the toxicity of ribonuclease molecules in order to select cytotoxic pathways on malignant cells. Therefore, it is necessary to discover the therapeutic potential and to study in detail the therapeutic values of ribonucleases.

Ribonucleases are potentially bioavailable drug candidates, and in regard to their toxicity, which is based on the selective hydrolysis of intracellular RNA molecules and specific cell membrane recognition processes, these enzymes can be used as anti-tumor drugs or therapeutic agents.

The importance of wild relatives in wheat breeding has caused a lot of investigations to study the genetic diversity and population structures in different species of wild wheat, using various molecular markers. The purpose of this study was to investigate the molecular diversity of 90 different accessions of Aegilops tauschiiand analyzing the genetic structure for clustering the populations using CAAT box-derived polymorphism (CBDP) markers.

In the present study, the genetic diversity and population structure of 90 different accessions of Ae. tauschii collected from different geographical areas of Iran, was evaluated using 12 CBDP primers.

Based on the results, the 12 primers amplified 141 bands in which 91 were polymorphic with an average of 7.58 bands pre primer. The polymorphism information content (PIC) and marker index (MI) for the CBDP primers ranged from 0.40 to 0.49 and 1.49 to 4.99, respectively. Primers CB9, CB12 and CB1 showed a high efficiency in genetic discrimination of evaluated accessions. Population structure analysis classified evaluated accessions into three main groups with 10, 18 and 30 accessions respectively and other accessions recognized as a mixture accessions. In this study, subpopulation NO.1 had the highest values of genetic diversity indices such as number of effective alleles (Ne), Shannon index (I) and heterozygosity (He). Among Ae. tauschii populations a very low genetic differentiation (Gst: 0.03) and high gene flow (Nm: 18.58) were observed. The dendrogram resulted from cluster analysis categorized the assessed accessions into three clusters which accordance with genetic structural analysis results.

Overall, the results of different parameters showed that the CBDP, is an appropriate marker system for assessing the genetic diversity of Aegilops tauschii accessions. Regarding to the results of genetic structural analysis and diversity parameters, a conservation program is recommended for management of Aegilops germplasm as a valuable genetic resource in the wheat breeding programs.

Leishmaniasis is one of the most prevalent parasitic unicellular diseases worldwide, which is common between human and animal. At present, antiviral compounds such as glucantime and amphotericin B are used to treat cutaneous leishmaniasis. In many cases, herbal remedies do not have any side effects and are both affordable and inexpensive. Therefore, finding a herbal medicine to treat leishmaniasis is one of the global goals. The aim of this study was to evaluate the anti-leishmanial activity of alcoholic extract of leaves of bay laurel (Laurus nobilis) on promastigote stage of Leishmania major by in vitro colorimetric method.

Different concentrations of leaf extract of bay laurel were studied against L. major promastigotes compared to amphotericin B. L. major promastigotes were cultured in RPMI-1640 medium supplemented with 10 Percent FBS. Optical absorption (OD) was measured by ELISA method to determine 50 Percent inhibitory concentration (IC50) of drugs. Promastigotes were added to the 96-well plate and incubated for 72 h with different concentrations of bay laurel leaf extract and amphotericin B (800, 600, 400, 200, 50, 25 and 10 μg ml-1). Flow cytometry was used to evaluate apoptosis. All concentrations were incubated in MTT assay for 3 min and flow cytometric for 2 h. Statistical data were analyzed by Duncan, ANOVA and SAS software at P≤0.05.

Maximum amount of apoptesis (97.35 Percent) in amphotericin B treatments as positive control was related to treatment 800 μg ml-1 concentration.  In leaf extract treatments of bay laurel, greatest amount of apoptosis (63.04 Percent) was related to treatment of 800 μg ml-1 concentration. CC50 of amphotericin B and leaf extract of bay laurel treatments was obtained in 129.6 and 200.5 μg ml-1 concentrations, respectively. Inhibitory concentration (IC50) of amphotericin B and leaf extract of bay laurel treatments was 18.5 and 589.5 μg ml-1 on L. major promastigotes.

The results were shown increased dose-dependent apoptosis based on flow cytometric.  The results of this study showed that proximity of L. major promastigotes and amphotericin B concentration treatments leads to apoptosis-like cell death, which increases with increasing drug concentration. According to the IC50 results of leaf extract of bay laurel and high its apoptetic efficiency, further studies are needed to evaluate its effective combinants of leaf extract on Leishmania parasite and also, to design clinical setting for this herbal drug.

Due to the low chilling requirement of almond, this plant encounters yearly with irreparable damages by late spring frost. The introduction of tolerant cultivars using genetic engineering methods, depends on the identification of molecular mechanisms involved in cold stress response. In recent studies the important role of small RNAs especially miRNAs have been confirmed in the response and adaptation of plants to biotic and abiotic stresses. Therefore, based on the sRNA sequencing results of almond, among identified cold-responsive miRNAs, in this study, the expression pattern of Pdu-miR319a and Pdu-miR398a-3p along with their targets were compared between two cold tolerant and sensitive genotypes of almond.

The reproductive tissues of almond (anther and ovary) from H as tolerant genotype and Sh12 as sensitive variety were treated under 0 and -2 ᵒC. After RNA extraction and cDNA synthesis Real Time PCR was performed and expression data were analyzed by 2- ΔΔct method.

MiR319a was identified with a positive regulatory effect in response to both cold stress levels in two genotypes. The downregulation of GAMYB-like was observed in both tissues of H under 0ᵒC and also in the ovary of Sh12 under 0 and -2 ᵒC. The expression of TCP4 was significantly increased in H under both stress levels. MiR398a-3p was detected with a negative regulatory effect in the H but the induction of CTR6 was observed in both tissues under -2ᵒC. In Sh12 the upregulation of miR398a-3p and the downregulation of CTR6 was observed.

Regarding to the differential expression of studied genes between two genotypes, in order to complete the molecular information about their function, it is recommended to examine their expression among other tolerant and sensitive varieties under different cold stress treatments, and followed by their function should be assessed using methods such as over expression, amiRNA technology and target mimicry.

The occurrence of planned cell death or apoptosis as a protected method is controlled by a number of genes that used to remove unnecessary cells. This cellular event is involved in immune and disease-related systems. Apoptosis is a cellular regulatory mechanism that balances the effects of cell proliferation and cell death. In the path of apoptosis, many genes and molecules are involved. Recently, developments using the RNAseq technique have shown that microRNAs play an important role in regulating planned cell death or apoptosis. The purpose of this study was to identify key genes and micro-RNAs affecting the apoptosis pathway in Androctonus crassicauda scorpions.

In this study, after transcriptome sequencing of venom glands of A. crassicauda scorpions using Illumina HiSeq 2000 platform and transcriptome assembly using Trinity software, the most important transcripts of the scorpion venom gland involved in apoptosis were identified using the KEGG database. Drawing of the apoptosis gene network with String software, analyzing the network of protein interactions and identifying key genes using cytoscape software and investigating the ontology of genes by the WebGestalt database were done. In addition, after identifying the scorpion microRNAs using homogeneous searches, the MRTarBase, TarBase, miRecords, and MirNet software programs were utilized to prediction of target genes and drawing a protein-microRNA interaction network.

Three pathways associated with apoptosis were identified, and 103 proteins were extracted from those. The results of protein network analysis revealed that 30 key genes involved in apoptosis were identified that according to obtained results, the most effective key genes involved in apoptosis are Akt1, bsk, Jra, Dronc, and rl. The results of statistical analysis of protein-microRNA interaction network also showed that mir-7-5p microRNA has the highest value.

The results of this study showed that microRNAs play an essential role in the regulation of many genes in the apoptosis pathway and can be used to regulate the expression of genes.

Environmental stresses affect the growth and development of crops during the growing season. Soil salinity is one of the most important environmental stresses that reduce crop yield. Understanding the mechanisms of coping with stress in plants at the molecular level can be effective in providing an efficient way to improve stress-tolerant plants. To save energy, plants do not express all their genes, simultaneously. Rather, depending on environmental conditions, they activate genes that are needed in a particular situation. For instance, in response to salinity stress, the expression of several genes in plants including HKT1 and NHX1 transporters is changed. In this study, variation in the expression of ZmHKT1 and ZmNHX1 genes along with the ZmMYB30 transcription factor were investigated in two salt-tolerant and salt- sensitive maize lines with real time polymerase chain reaction technology.

Maize salt-tolerant line (P14L2) and salt-sensitive line (MO17) seeds were grown in plastic pots containing perlite and peat moss in 2:3 ratios under controlled conditions in growth chamber, and Hoagland solution was used to irrigate plants. In the eight-leaf stage, salinity stress from NaCl source was gradually applied to half of the pots starting from a concentration of 5 ds/m for three days, and from the fourth day onwards, salt stress was applied at a concentration of 8 ds/m for 7 days. The other half was used as control. Then, 24 hours (as short-time salt stress) and 7 days (as long-time salt stress) after the salinity stress, the roots and leaves of the plants were sampled in liquid nitrogen. RNA extraction was performed from leaf and root samples, and cDNA was then synthesized. Gene expression was assessed with real-time PCR. The Actin gene was used as the reference gene. The experiment was performed in 2 biological (experimental) and 3 technical (laboratory) replicates. Finally, the changes in the expression of genes were measured.

Increased expression of ZmNHX1 gene in root tissue during the short-time and in leaf tissue during the long-time in the tolerant line, and also increased expression of ZmHKT1 gene in the root tissue during the long-time in the tolerant line compared to the sensitive line probably indicate a positive role of these genes in resistance to salinity stress in maize. The highest relative expression of MYB30 gene in both tolerant and sensitive lines was related to root tissue during the short-time, which over time a significant decrease in the expression of this gene was observed in both lines.

The results of this study can be potentially useful in maize breeding programs to produce salinity resistant cultivars.

Late embryonic accumulating proteins (LEAs) are proteins that involved in reducing Cellular injuries associated with dehydration and preventing cellular proteins from aggregation due to osmotic stress and freezing. Increased expression of some genes encoding these proteins has led to increased resistance to stresses such as dehydration, cold and salinity in many plants. Analysis of biological data with the using of bioinformatics tools plays an important role in the study of genes and proteins and predicts their function in response to the stress.

In this study, in order to study of LEA proteins in two important grasses (tolerant and sensitive to drought), the interest sequences are obtained from different data bases and the phylogeny tree are drown using ClustalW software. After the investigation of classes in different groups, the short sequences deleted and some sequences were selected. The information about sequence characteristics, their motifs, prediction of intracellular location, analysis of hydrogen and non-hydrogen bands and prediction of biological process and molecular activity obtained using ProtParam, EXPASY, MEME and SMART, Wolf PSORT, PIC, WHAT IF databases and Blast2GO software. The 3D structure of these proteins was modeled using Discovery studio, HyperChem, MODELLER v10 and SPDBV software.

The results were grouped the proteins into seven distinct groups with the highest number of sequences in dehydrin group. Most proteins had small size with less than 30 kDa. Also, the most sequences showed high surface charge, because of their hydrophilicity and flexible structure to form a chaperone structure to protect the cell membrane from stress. The study of intracellular location showed that most of proteins are located in the cell nucleus. Also, the motif study demonstrated the conserved sequences among members of the same group. The prediction of molecular activity and biology indicated the role of these proteins in the tolerance to the stress including drought and protein folding through bindings or catalytic activity.  

Barley as a stress-resistant plant mainly involved the dehydrin sequences and rice as a stress-sensitive plant mainly contains the LEA4 and LEA2 sequences.

Temperature is a critical variable in health and disease. Thermal stressors such as heat stress elicit neuroendocrine responses including activation of the HPA axis .Heat stress is a challenge for animal and human society. It has been shown that brain monoaminergic system is changed during acute and chronic heat stress condition. Commercial and domestic poultry are very susceptible animals to the heat stress. High environmental temperature led to heat stress condition and had negative effect on physiological performance. One effect of heat stress is changed molecular and cellular mechanisms. These changes in diencephalon, as an awaking center (thalamus) and autonomic mechanisms center (hypothalamus), have profound physiological performance results. However the goal of experiment was to investigate effect of heat stress on diencephalic BDNF gene expression and blood corticostrone level.

In this experiment, 20 seven-day-old male chicks divided in 1- heat stress group (10 chicks) and 2- control group (10 chicks in normal temperature condition). Heat stress induced by 39 C◦ and continued to 5 hours. Control group temperature was 30 C◦. At the end of 5 hours duration, chicks was anesthetized with Isofluran gas and killed. Blood gathered from jugular vein. Brain dissected and diencephalon was frozen in liquid nitrogen immediately. Blood corticosterone level was measured by ELIZA method, BDNF gene expression assayed by quantitative real time PCR technique with the SYBR green method.

In this experiment, heat stress significantly reduced diencephalic BDNF gene expression. However, corticosterone level significantly elevated in heat stress exposed chicks. It would be able to say that there is a relation between BDNF gene expression and blood corticosterone concentration.

These results showed changes in BDNF gene expression during heat stress condition is associated to blood corticosterone level elevation. Taken together, the results showed that a significant relationship between Hypothalamus-Hypophysis- Adernal cortex activity during acute heat stress condition and diencephalic BDNF gene expression as a neuroprotective agent of brain.

Raini Cashmere goat is one of the most important goat breeds in Iran. These animals are breed both for meat production and cashmere production. One of the basic measures in the domestic animals is the study of genes and proteins associated with economic traits and their study at cellular or chromosomal levels. One of these important genes is the leptin. Leptin is produced by white adipose tissue and plays an important role in the regulation of feed intake, energy balance, fertility and immune functions. The aim of this research was to study leptin gene expression in adipose tissue, liver, kidney, lung and heart of Raini Cashmere goat using Real Time PCR technique.  

Tissue sampling from heart, lung, liver, kidney and adipose tissue (3 replicates from each tissue) of 6 animals was performed and RNA was extracted. Extracted RNA were immediately stored at -80°C.The Quality and quantity of RNA were evaluated and cDNA was synthesized and Real Time PCR was performed. PCR Products were electrophoresed on 1.5% agarose gel. Melting curves from Real Time PCR were examined and were evaluated different levels of expression in the studied different tissues.

The results of Real Time PCR curves and observation of electrophoresis of PCR products on agarose gel showed that the leptin gene was expressed in all the tested tissues and the highest level of expression was observed in adipose tissue (4.5) and liver (3.7) and the lowest level was detected in heart (1.4).  

These results may show that leptin plays a particular role in fat metabolism. Further studies are needed to clarify role of leptin in the physiology of fat metabolism and other materials. This would help us to better understand the mechanisms for the known effect of nutritional factors and body fatness on various functions.