مجله بیوتکنولوژی کشاورزی
سال چهاردهم شماره 2 (پیاپی 47، تابستان 1401)

The use of plant expression systems to produce recombinant proteins have advantages such as different methods for transformation and culture, occurrence of appropriate post-translational modifications and no risk of contamination with animal and microbial pathogens. However, low productivity in these expression systems have always been a major challenge. Secretion-based expression systems, such as Hairy Root Cultures (HRCs), is a convenient way to continuously produce active ingredients without the need for plant tissue degradation, which will facilitate the purification process and significantly reduce related costs. This study aimed to designing and constructing vectors carrying NtREL1 root specific promoter in combination with patatin, pectin methyl esterase and calreticulin signal peptides at the same time in order to increase the specific expression of recombinant proteins in hairy root culture and their subsequent secretion into hydroponic medium.

The nucleotide sequence of NtREL1 root specific promoter, patatin, pectin methyl esterase and calreticulin signal peptides were retrieved from NCBI database. To identify cis acting regulatory elements involved in specific expression, the NtREL1 sequence was investigated by PLACE and PLANT CARE softwares. Bioinformatics studies were performed to design pBI121 expression vector with NtREL1 promoter in combination with signal peptides and appropriate restriction sites. After receiving the synthetic fragments in pUC57 vector, the fragments were digested by restriction endonuclease and sub cloned in pBI121 expression vector.

Examination of the NtREL1 promoter showed that this promoter is rich in AT nucleotides. These sequences can improve the recognition process by transcription systems and increase gene expression due to their ease of conversion from double-stranded to single-stranded form. The accuracy of NtREL1 promoter sub cloning upstream of patatin, pectin methyl esterase and calreticulin signal peptides in pBI121 expression vector were confirmed using colony PCR, digestion reactions and sequencing.

Since pBI121-based expression vectors are more efficient than other expression vectors and widely used in genetic transformation and expression of recombinant proteins in plants, this recombinant vectors can be applied effectively for recombinant protein production and its secretion in expression systems like hairy root culture.

Hepatitis C infection is one of the most common chronic infectious diseases in the world and about 3-5 million people are infected with this infection annually. Unfortunately, despite recent advances, there are still widespread problems for the treatment of these people and many needs have not been met. As a result, there is an urgent need for more effective treatment strategies to treat this disease. One of these new therapeutic approaches is the use of recombinant proteins, which have no side effects for the patient and at the same time are cheap and available.

To conduct this study, the three-dimensional structures of virus proteases (NS2, NS3, NS4A, NS5A and NS5B) and recombinant peptide CLF36, were obtained. The chemical structure of new generation drugs was also downloaded from PubChem. To evaluate the stability of the predicted structures and to ensure the accuracy of the third structure of proteins, molecular dynamics simulations were performed using GROMACS software version 2019 and in a duration of 10 nanoseconds. Then, to evaluate the antiviral properties of the peptide, molecular docking was performed for peptides and drugs under static conditions. To evaluate the stability of the complexes, based on the results obtained from molecular docking, complexes with negative values of free binding energy were selected to perform molecular dynamics simulations. Finally, the obtained results were evaluated under dynamic conditions in GROMACS software. The engineered peptides were then designed according to the results and the sequence of the best engineered peptides was sent for synthesis. After cloning and expression of the recombinant peptide in the yeast host, the IC50 level of the peptide on the Huh7.5 liver cell line was determined and the virus-infected cells were treated with the desired peptide. After extracting RNA from the cells, the virus loaded in the sample. Were measured.

Among the results obtained from various dockings performed between virus proteases and recombinant peptides and commercial drugs, it was found that the peptide-binding strength (average -99.92) was much higher than other drugs (average -61.54). This indicates the high antiviral property of the recombinant peptide. The results of molecular dynamics showed that the values of rmsd and hydrogen bonds and binding energy for CLF36 peptide bound to virus proteases compared to other drugs indicate a stronger binding of this peptide. In addition, the engineered peptide showed stronger antiviral activity compared to the natural peptide (binding energy was 8 units stronger). Examination of the function of the peptide in vitro also showed that this peptide has a stronger antiviral effect compared to drugs in that it was able to completely prevent the virus from multiplying in cells.

The final results of this study indicated that recombinant peptide, has strong antiviral properties. However, to confirm these results, additional laboratory studies are needed.

The aim of present study was transient expression and purification of recombinant caprine chymosin and prochymosin in N. benthamiana leaves using plant binary vectors and tobacco mosaic virus (TMV) vector.

The caprine prochymosin gene sequence was obtained from NCBI gene bank and optimized at codons for preferential expression in tobacco leaves. The chymosin and prochymosin genes were introduced into the plant binary and viral vectors. Immunoblotting analyses were conducted to demonstrate expression of chymosin and prochymosin, and the expression level was quantified by milk clotting activity test.

Results indicated that N. benthamiana leave cells cannot process prochymosin correctly and only a weak band observed in extracted total proteins (TPs) which showed no milk clotting activity while leaves infiltrated by p20αchymosin showed a band with chymosin molecular weight and slightly showed milk clotting activity. TMV-based recombinant prochymosin and chymosin expression showed necrosis after 3 days post infiltration (dpi) in agro-infiltrated leaves and after 5 days, leaves completely dried and necrotized. Blotting analysis showed only a band with right molecular weight in TSPs extracted N. benthamiana leaves infiltrated by TMVαchymosin. Moreover, we subcloned chymosin which expanded with a N-terminal barley alpha amylase signal peptide and a C-terminal hybrid Fc tag which labelled as TMVαchymosinhyFc. The expression level increased gradually from 2 to 5 days after infiltration from 3.3 U/g FW to 16.8 U/g FW but after 5-day leaves necrosis started and expression level reduced gradually.

Viral vectors and transient expression can be a cheap, fast and effective method to produce a huge amount of recombinant proteins but it seems chymosin production still needs more research and find the N. benthamiana cells problem with chymosin which leads to necrosis and reduce the expression level for large scale production.

The aim of this study was to determine the relationships and genetic diversity between and within wild species of Aegilops using EST-derived TRAP molecular markers.

In this study, genetic diversity and relationships among eight wild species of Aegilops collected from 14 provinces of Iran were evaluated using 24 TRAP primer combinations.

The mean of PIC for studied primers was 0.95 and the lowest and highest values ​​of this index were related to TRAP20 (0.92) and TRAP10 (0.97), respectively. Also, the mean of MI was 10.77 and the lowest and highest values ​​were related to TRAP3 (6.95) and TRAP17 (17.34), respectively. The molecular analysis of variance showed 60% variance between species and 40% variance within species. Estimated genetic parameters showed that the highest levels of Na (1.07), Ne (1.26), I (0.23) and uHe (0.16) belonged to Ae. truncialis and the maximum Nei gene diversity (0.14) was related to Ae truncialis, Ae. umbelulata and Ae. neglecta. Minimum values ​​of Nei gene diversity (0.09), Shannon index (0.14), No. of effective alleles (1.16) and No. of observed alleles (0.79) was observed for Ae. crassa. The least genetic similarity was observed between Ae. crassa with Ae. cylandrica and Ae. umbelulata and the most similarity between Ae. triuncialis and Ae. umbelulata. Being in the same group of Ae. umbelulata and Ae. truncialis with cluster analysis and PCOA confirmed the high genetic similarity of these species.

The results showed high genetic diversity among and within species. Molecular analysis of variance revealed higher genetic diversity between species, indicating low gene flow and Fst, as well as a high genetic differentiation between species.

Foot-and-mouth disease (FMD) is an acute highly contagious viral disease in susceptible cloven-hoofed animals, which causes extensive economic loss in livestock industry. Recently, diagnostic methods have been developed to detect antibodies against non-structural proteins of this virus. Non-structural proteins 3ABC and 3D that provide linear B cells epitopes allow effective detection of virus-infected animals. In this study, immuno-dominant epitopes of 3ABD proteins were considered for the synthesis of a new gene sequence and assembling of 3AB and 3D fragments.

Overlap extension-polymerase chain reaction (OE-PCR) was used to synthesize 3ABD antigenic sequences. Viral RNA was isolate from BHK-infected cells and reverse transcribed. In order to remove 654 nucleotides of the middle 3C genomic sequence, a pair of internal primers with 29 overlapping nucleotides and two external primers were designed. The 3AB and 3D fragments were each amplified in separate PCR reactions and purified from the gel after electrophoresis. These fragments were used as templates in a one-step OE-PCR for the synthesis of 3ABD sequence by external primers and Pfu DNA polymerase.

The results showed amplification of 3AB (418 bp) and 3D (557 bp) fragments with overlapping sequences at the end of 3' and 5', respectively. The OE-PCR experiment resulted in amplification of multiple fragments, and the 3ABD fragment with the desired length (946 bp) was confirmed after sequencing. Only by considering 29 homologous nucleotides in the design of overlapping primers and a high-fidelity, highly accurate DNA polymerase, two epitopic regions of non-structural proteins of FMD virus (3'-3AB and 5'-3D) were linked together.

The new gene sequence synthesized by a one-step OE-PCR technique has the antigenic sites of 3ABD non-structural proteins of FMD virus, obviates the need for expensive peptide synthesis, and can be a proper alternative for production of 3ABD recombinant protein for application in detection of FMD by Enzyme Linked Immunosorbent Assay.

Sugarcane (Saccharum officinarum L.) is a commercial plant that extracts more than 70% of the sugar produced worldwide. Salinity in many arid and semi-arid regions of the world is considered as a major problem and limiting factor for growth, quality and yield of crops including sugarcane. Chromosome manipulation of the plant species is one of the most powerful methods of plant breeding because of its effect on some morphological, physiological and genetic variation. The aim of this study was to investigate the effect of different levels of colchicine mutagen on changes in DNA content and to investigate the effect of increasing DNA content on the transcription rate of catalase gene.

In this experiment, sugarcane seedlings were treated with colchicine (concentrations of 0, 100, 200 and 400 mg / L) in two CP48 and CP69 cultivars for 8, 24 and 48 hours, respectively. The plants were then examined for morphological and physiological characteristics. Plants were then exposed to saline stress (200 mM NaCl) and normal. After applying stress, plants with probable polyploidy were examined for genetic content using flow cytometry. Also, to investigate the expression pattern of Catalase gene (CAT2), leaf sampling was done 24 h after salinity stress from control and stressed plants.

The results showed that viability, height and density of stomata decreased with increasing colchicine concentration and duration of treatment. However, stomata size increased with increasing colchicine concentration and duration of treatment and chlorophyll and photosynthesis increased with increasing duration of treatment. The results showed that the expression of this gene was significantly increased in both CP48 and CP69 cultivars under salinity stress. The expression of this gene was increased in CP69 cultivar treated with colchicine and this increase was significant at 200 mg / L colchicine. While in CP48 cultivar the effect of colchicine treatment was not significant.

Based on the above results, it can be concluded that increasing the genetic content of sugarcane can improve salinity resistance.

Different climates and wide geographical area of Iran have caused considerable genetic diversity in Iranian chicken ecotypes. Artificial selection for genetic gain in economic traits leads to a reduction of genetic diversity in livestock and poultry breeds. On the other hand, by identifying the genetic potential of native ecotypes and managing breeding programs, it is possible to increase productivity in native chicken populations while maintaining genetic diversity. However, so far no genomic study has been performed to identify the genetic characteristics of native chickens. The aim of this study was to identify the genetic potential of Iranian native chicken ecotypes for appropriate targeting of breeding and genetic conservation programs.

In this study, genomic data related to 51 native chicken ecotypes, 11 chickens of Arian line and 10 chickens of Leghorn breed were collected from the department of animal science at Shahid Bahonar University of Kerman. Mapping step against reference genome was done by BWA program. Identification of single nucleotide variants was performed by GTAK software. Phylogenetic analysis was investigated using the neighborhood joining method and Mega software. Fst and inbreeding coefficient among population were performed using Admixture and VCFtools programs. 

Percentage of alignment against the reference genome was reported between 83% and 95% for all samples and also 14.56 million single nucleotide variants were reported from the native and commercial chicken genomes. The results of phylogenetic tree analysis showed that almost all studied ecotypes were classified into separate groups. According to the reported results, it can be claimed that native chicken ecotypes are more genetically similar to the Arian line (compared to Leghorn breed). Findings obtained from admixture and Fst analysis also confirmed the results of phylogenetic analysis. For example, the highest Fst was estimated as 0.219 between Leghorn breed and Marandi ecotype.

Due to the genetic similarity of most native chicken ecotypes and Arian line, breeding programs can be organized based on meat production traits. By targeting breeding programs, genetic resources can be improved to increase productivity while preserving the genetic diversity of native chicken ecotypes.

Orchard Grass (Dactylis glomerata L.) is a perennial forage and cross-pollinating grass, has wide genetic distribution in Iran. Although the genus Dactylis has been studied quite well within the past decades, little is known about the genetic diversity and population patterns of natural populations in grassland regions. The aim of this study is to identify the genetic diversity of this species in the East Azerbaijan, Iran.

In this study, seeds of 25 ecotypes of Dactylis glomerata L. were planted in the research farm of the Agricultural Biotechnology Research Institute of Iran, Northwest & West region in Tabriz as a randomized complete block design with 3 replications. Then 34 selected genotypes (single plant) were examined using 22 ISSR markers.

According to the results of ISSR markers, a total of 424 loci were amplified in the genome, of which 34 loci among all studied genotypes were monomorphic and 390 loci were polymorphic. The mean percentage of polymorphisms for D. glomerata L. in ISSR markers was estimated to be 70.39%. Also, the mean PIC value (polymorphic information content) for ISSR markers was equal to 0.288, the minimum PIC value for ISSR14 marker (0.175) and the maximum PIC value belonged to ISSR22 marker (0.341). Grouping of D. glomerata L. genotypes based on ISSR markers was performed using Mega 4.0.2 and Structure software, which grouped the results of Mega 4.0.2 genotypes into 4 groups, and Structure software grouped the genotypes into 2 groups.

The results indicate that the using of ISSR markers to differentiate close kin populations has a high advantage and plays a significant role in the differentiation of individuals. Also, the high allelic diversity of ISSR markers indicate a large level of diversity in D. glomerata L. populations and it’s a suitable tool for studying the genetic diversity of D. glomerata L.

Fennel in animal diets for various purposes including improving weight gain, oxidative quality of meat, digestion and growth efficiency, closed cell volume, small intestine length and weight, carcass efficiency, feed intake, red blood cells and hemoglobin count, feed conversion, efficiency and health status and reduction of the total number of bacteria have been used. On the other hand, DLK1 gene plays an important role in controlling various processes throughout the fetus and adulthood. Therefore, the aim of this study was to investigate the effect of fennel on DLK1 gene expression in Kermani sheep heart using real-time PCR.

In this study, 30 male Kermani lambs (6-month-old) with almost the same weight were selected and randomly divided into three experimental groups (10 in each group). Experimental animals were fed freely and separately with three levels of fennel (0, 1 and 2%) for three months. After slaughtering, the heart weight and fat around the heart were measured and the heart tissue was sampled. RNA extraction and cDNA synthesis were performed using standard kits. The expression of DLK1 genes (target gene) and beta-actin gene (reference gene) was performed using proper primers using real-time PCR method and the results were analyzed with the appropriate software.

The results of quality assay of extracted RNAs on agarose gel and at A260 / A280 wavelength showed good quality of total RNA. The results of real-time PCR and electrophoresis of PCR products on agarose gel showed that DLK1 gene was expressed in heart tissue. Adding fennel to the diet did not significantly change the weight of the heart and the weight of fat around the heart compared to the control diet. The results showed that fennel nutrition at 1% and 2% levels significantly (P <0.05) increased DLK1 gene expression in heart tissue.

Based on the results, it can be concluded that fennel can be used in sheep diet to improve heart structure through positive effects on DLK1 gene expression. Since fennel has increased the expression level of DLK1 gene in heart tissue, it can be considered to improve heart function. It can be concluded that fennel can be used for different purposes in livestock, but for each effect in each tissue, more research should be done considering different genetic, epigenetic and physiological conditions to reach the distinct conclusion.

Honeybees, as pollinating insects, are an important part of nature. Because behavioral traits are so important in honeybees, comparing brain tissue transcriptomes of the two subspecies with aggressive and calm behavioral characteristics makes it possible to understand this behavioral difference genetically. This study aimed to investigate to gene expression profile and identify the key genes in brain tissue in Italian (Apis Mellifera Ligustica) and African (Apis mellifera Scutellata) honeybees concerning behavioral traits. The Italian honeybee has calm behavioral characteristics, while the African is known as an aggressive honeybee.

RNA-Seq data were obtained from the NCBI  (GEO) database, and after pre-processing of reads, the brain tissue transcriptomes of both subspecies were aligned and mapped on the honey bee reference genome (v A.mel 4.5), and then data qualification, transcriptome assembly, differential expression analysis, and gene ontology were performed.

Differential gene expression analysis identified 16,701 genes on the honeybee reference genome, of which 22 genes in brain tissue between the two subspecies had significant differential expression (adj p-value <0 .05 and Log2FC>2). As well, some of these genes were first identified. Gene ontology analysis showed that among these 22 genes, such as ITPR, MRJP, HSP70Ab, MBS, GB45410, and Def1 are directly or indirectly involved in the occurrence of various traits such as defensive, health behavior, reproductive, heat, light, and smell sensitivity. In addition, the SNPs encoding the honeybee brain tissue genome were identified in both subspecies, and 99636 SNPs were identified in the Italian, and 92514 SNPs were identified in the African subspecies.

RNA-seq data, due to its high throughput, can provide us with accurate information about the expression of genes in different tissues in various subspecies. In this study, genes involved in honeybee behavioral traits and the SNPs in these genes were identified

The growth and yield of plants in the arid and semiarid areas are limited by salinity stress. In these areas, salinity intensifies by high temperatures. Peppermint (Mentha piperita L.) is of great economic importance in the most of world areas due to their valuable essential oils. This study investigated the simultaneous effects of salinity and heat on rosmarinic acid production and expression of three effective genes (C4H, HPPR, and RAS) in rosmarinic biosynthesis in peppermint.

The effect of two levels of salinity 60 and 120 mM of sodium chloride (irrigation water with salinity of 10 Mm as control) and heat stress at 35°C (25°C as control) at three interval times (24, 48 and 72 hours) after treatments were measured in a completely randomized design with three replications.

The results showed that salinity stress decreased the relative level of rosmarinic acid. The process intensified over time so that it reached its lowest level at salinity of 60 mM at 72 hours. RAS genes expression was significantly reduced under salinity of 120 mM and 60 mM under 25˚C and 35 ˚C. Changes in the expression level of C4H in peppermint seedlings at the salinity levels of 60 mM and 120 mM at 25˚C showed a significant increase (4.1 and 3.6 times respectively) at the first 24 hours compared to control. The expression of HPPR gene in peppermint seedlings at 35 ° C increased 2.68, 1.79 and 2.55 times at 24, 48 and 72 hours, respectively, compared to the control.

Simultaneous salinity and heat stress are a limitation factor for growing Mentha piperita L. These factors had a negative effect on the biosynthesis of rosmarinic acid in this plant. Salinity and heat stress decreased the expression of some genes and increased the expression of others in peppermint.