مجله بیوتکنولوژی کشاورزی
سال سیزدهم شماره 4 (پیاپی 45، زمستان 1400)

Geranium is one of the most economically important pot plants in the world and is widely used in landscape. In order to produce disease-free and homogenic plants, proliferation by tissue culture is one of the alternative methods for mass production of this valuable plant. The aim of this study was to achieve an efficient and rapid method for micropropagation of Geranium by comparing leaf discs, petiole, stem and root explants on MS and B5 media.

For callus induction, stem, leaf and root explants were cultured on MS basal medium with 11 different kinds of hormonal combination, containing auxin (NAA and IAA) and cytokinin (BAP, Kin and Zeatin) with various concentrations. For direct regeneration of stem, leaf and petiole explants, 10 different culture media were used in MS and B5. After shooting, the explants were transferred to B5 culture medium containing auxin (0.5 mg/l NAA and 0.5 mg/l IAA) for root induction.

The best hormonal treatment for callus induction was 1mg/l NAA+10mg/l Kin by using stem and leaf explants as 92.5% and 88.75%, respectively. Also, the maximum fresh and dry weight of callus were related to stem explants on MS culture medium included 1mg/l NAA+10mg/l Kin, which were not significantly different from leaf explants on the same culture medium. The maximum percentage of direct regeneration and number of branches, respectively as 71.25% and 7.12 shoots, were related to stem explant on B5 medium containing 2mg/l BAP+1mg/l Zeatin, and the maximum shoot length, as 2.56 cm, was observed during direct regeneration in MS culture medium included 0.2 mg/l NAA+0.5 mg/l BAP+1 mg/l Zeatin. The shoots were successfully rooted and adapted to the environment.

The results of the present study were showed that the micropropagation of ornamental geranium plant is affected by the type of explant, the concentration of growth regulators and the type of culture medium and the stem explant is efficient in callus induction and direct regeneration.

Brucellosis is a common disease between humans and livestock that has caused a great deal of damage to the livestock industry in recent years. One way to prevent this disease is to get a vaccine to immunize disease-prone herds. To produce a successful recombinant vaccine, factors such as: selecting the right adjuvant, selecting the right antigen and the right delivery system are very important. To this end, epitopes have recently been widely used in clinical research, biomedicine, and the production of recombinant vaccines. Surface proteins of this bacterium, including BLS and Omp25 proteins, have antigenic properties and play an important role in causing this disease. Due to the lack of effective vaccines, today the need to produce new and effective vaccines is felt more than ever. The aim of this study was to bioinformatically predict the epitopes of BLS and Omp25 genes of Brucella bacterium in order to introduce a suitable candidate for vaccine production.

For this purpose, SYFFPEITHI, PROPRED and IEDB servers were used to predict epitopes. Since the three-dimensional structure of the protein is required to investigate the interaction of antigens with cell surface receptors, in the next step, the predicted epitopes were modeled using the Pepfold 3 server. In addition, in order to evaluate the stability of the predicted structures and to ensure the accuracy of the third structure of the peptides, molecular dynamics simulations were performed using GROMACS software version 2019 and in a duration of 10 nanoseconds. Then, the three-dimensional structure was obtained by modeling homology method and the docking study of the interaction between epitopes with sheep MHCII and MHCI cells was performed using Autodock vina software.

Then, in order 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, 8 peptides with better numbers were considered as suggested epitopes. The results obtained from molecular dynamics showed that the rmsd values for interleukin and interleukin bound to the polypeptide did not differ much in the two cases, so these results indicate that the activity of interleukin bound to the polypeptope did not change. The rmsf values also indicate the same stability in both proteins.Finally, the obtained results were evaluated in dynamic conditions in GROMACS software. The results of this study showed that in dynamic conditions, 4 predicted epitopes have high binding power to MHC class 1 and 2 receptors.

 In addition, docking and molecular dynamics results showed that the polypeptide predicted in this study could be used as a candidate polypeptide in the design of a recombinant vaccine against brucellosis. However, to confirm these results, additional and laboratory studies are needed.

Proteases are among the most important industrial enzymes. Microbial proteases, especially from Bacillus sp., are most widely exploited industrially. The aim of this study was isolation, cloning, sequencing, expression, and bioinformatics study of yyxA serine protease gene extracted from Bacillus licheniformis.

In this study, after extraction of bacterial DNA, the yyxA serine protease gene was isolated from Bacillus licheniformis using the polymerase chain reaction technique and cloned into the pTG19-T vector. The molecular structure, its biochemical and phylogenetic properties were investigated. The three-dimensional structure of the cloned enzyme was predicted using the I-TASSER, PHYRE2, RAPTORX, and Modeller tools. Confirmation of yyxA gene expression was performed by SDS-PAGE and dot blot analysis.

Cloning was confirmed by sequencing. Based on the results of phylogenetic studies, the obtained protein sequence showed high similarity to the sequences of other Bacillus species, such as B. subtilis, B. gobiensis, and B. pumilus. After evaluating the drawn models, it was found that the models provided by PHYRE2 and I-TASSER software were desirable ones for predicting the three-dimensional structure of this protease. Recombinant protein production was successfully induced by IPTG induction in the host containing the plasmid pET28a-yyxA. Optimization of recombinant protein production was investigated. The highest expression values were obtained at 37 ° C for 4 hours with one mM IPTG.

The nucleotide sequence of the yyxA gene is 1212 nucleotides long, encoding a protein with 403 amino acids. Studies have shown that the enzyme encoded by this gene is in the category of stable enzyme and will be expressed in solution in Escherichia coli. These advantages make the enzyme as a suitable candidate for use in industry.

Plants are very efficient in producing valuable pharmaceutical proteins. Human epidermal growth factor (hEGF) has numerous effects on various cellular systems, including wound healing, organogenesis, and so on. The present study was carried out with the aim of constructing monocotyledonous-specific vectors for hegf insertion and mass-production in a self-pollinated crop, barley, afterwards. This crop plant, with high protein yield and very low cross-pollination, is an ideal host for the production of epidermal growth factor.

The hegf sequence was optimized by COOL software, based on the codon preference of the host plant, and synthesized with a specific promoter, D-hordein. The encoding sequence of signal peptide targeting protein storage organelles in barley seeds was included at the beginning of the encoding area. The synthesized gene cassette was isolated from the cloning vector pUC57Hvorhegf by SacI and HindIII and cloned in pBI121. To insert the gene cassette of interest into pGH215, due to the lack of similar restriction sites in pUC57Hvorhegf and pGH215, the intermediate vector, pBI121Gus-12, was applied. After digesting pUC57Hvorhegf and pBI121Gus-12 by SacI and KpnI, the sequence of interest was incorporated into pBI121Gus-12. Finally, the recombinant pBI121Gus-12 and pGH215 were digested with KpnI and SalI, and the gene cassette was cloned at the right border of T-DNA, behind the NOS terminator.

Cloning accuracy and constructing pBI121Hvorhegf and pGH215Hvorhegf vectors bearing kanamycin and hygromycin, respectively, suitable for monocotyledons-crop transformation was confirmed by colony PCR, digestion pattern, and sequencing with forward and reverse primers.

The modified recombinant expression vector pGH215 with the gene of interest carrying the hygromycin gene cassette, giving a high level of ability to select transformed explants, can be used to transfer desired genes in monocotyledons and express those genes in protein storage organelles.

Alfalfa mosaic virus (AMV) is one of the most important plant viruses that has a wide host range and economically is a destructive virus in the world and Iran. The aim of this research is to study the MP gene of Iranian AMV isolates and its application in the phylogenetical analysis and compare it with the CP gene. 

During 2014 to 2017, a number of plant samples including alfalfa and weeds were collected from alfalfa fields in six Iranian provinces. ELISA test using polyclonal antibodies and RT-PCR were employed to check the AMV infection of the samples. Among the AMV infected samples, 20 isolates including 17 from alfalfa and three wild species including Sonchus oleraceus L., Chenopodium amaranticolor L. and Plantago ovate L. were chosen for phylogenetical analysis based on the sequence of the MP gene. 

Phylogenetic analysis based on the nucleotide sequence of movement protein (MP) gene of AMV including Iranian (n=20) and GenBank isolates (n=15) showed that all isolates are divided into two group I and II and each group is also divided into two subgroups A and B. The majority of Iranian isolates were placed in group II. All abroad and two Iranian isolates (Accession numbers: KX535454 and KX535456) were placed in subgroup IA, and subgroup IB is limited to three Iranian isolates (Accession numbers: KX535460, KX535458 and KX535462). Whereas based on the part of the nucleotide sequence of the MP gene (468nt), most of Iranian isolates, together with one Spanish isolate (JQ691163) were clustered in a new subgroup (IIB). 

According to the results of this study, it seems that the MP gene can be used to analyze the phylogenetic relationships between AMV isolates. Since the origin of AMV is from Far-East and central Asia and due to the host variability of the virus in these regions, AMV probably has a high genetic diversity in Iran.

This study was conducted to investigate the application of the start codon targeted (SCoT) markers in the genetic diversity of tomato. In addition, the genetic diversity in important cultivars of tomato, which have been previously cultivated, are being cultivated, or are hoped to be cultivated in Iran were assessed.

Ninety nine 99 tomato cultivars were investigated for SCoT polymorphism. DNA isolation and SCoT analysis were carried out using the fresh leaf samples. Thirty-six SCoT primers were initially screened for analysis and fifteen primers were considered for the further analysis.

The cultivars produced 207 amplicons while the 206 were polymorphic. Amplicon size varied from 250 to 3200 base pair (bp). The average of polymorphism and the mean of polymorphic information content were 99.52 and 0.30, respectively. Also, Jaccard's similarity coefficient was applied and the mean Jaccard genetic similarity coefficient was 0.52. According to Jaccard's coefficient, the lowest similarity (0.17) has been observed for cultivars 34 and 97 while the highest similarity (0.84) were detected between cultivars 86 and 87. In addition, the cluster analysis was conducted based on Jaccard similarity coefficient and centroid method which classified cultivars into three clusters. Besides, the principal coordinate analysis was considered. Accordingly, the cultivars divided into four groups and the first three components explained 58.82% of the molecular variation. The results of the principal coordinate analysis were largely consistent with the results of the cluster analysis. Having high polymorphic information content, marker index, effective multiplex ratio, and resolution power, SCoT12 and SCoT23 primers were properly effective in differentiating the cultivars.

This study showed that SCoT molecular markers are appropriate for investigating the genetic diversity amongst tomato genotypes and generate a high level of polymorphism. Thus, these markers have considerable efficiency in differentiating tomato genotypes. In addition, this investigation indicated that SCoT12 and SCoT23 primers were suitably effective in differentiating the cultivars. Furthermore, our resech claimed that tomato cultivars that are used in Iran do not have high genetic diversity while for more precise conclusion; it is suggested to practice more SCoT primers along with other markers.

Soybean (Glycine max L.) is one of the most important agricultural products that is of special importance not only because of its capacity in oil production but also because of its role in human and animal nutrition. One of the effective methods in overcoming the breeding problems of this crop is the use of in vitro culture techniques and molecular methods.  The use of plant tissue culture techniques is one of the effective methods in overcoming the breeding problems of this crop. Tissue culture and plant regeneration in the handling process depend on several factors. The aim of this study was to optimize the effective factors such as genotype, type of micro sample, type of gelling agent and combination of growth regulators in soybean regeneration. 

After germination and seedling growth of Saman and DPX cultivars in culture medium, hypocotyl and cotyledon micro samples in culture medium containing BAP hormone treatments (0, 1 and 2 mg/l) And IBA (0, 0.1 and 0.5 mg/l) were cultured. Subsequently, the samples were recovered in culture media containing IBA (0, 0.5, 1 and 1.5 mg/l) with agar (5, 6 and 7 g) or gellate (2 and 3 g) were cultured. Separately, the effect of growth regulators on soybean cultivars was performed as a CRD-based factorial experiment, the effect of gelling agents on organogenesis and rooting experiment of cultivars was performed in a completely randomized design with three replications and each replicate containing six samples. 

The results of this study showed that in the cotyledon sample of Saman cultivar under hormonal treatment of 2 μg/l BAP and 0.1 μg/l IBA, the highest rate of organogenesis and number of regenerated branches were observed. Also, the presence of 7 g of agar in the culture medium showed the highest rate of organogenesis and the number of regenerated branches. In addition, a concentration of 1.5 micrograms per liter of IBA induced the highest rooting rate. 

Saman cultivar showed better results in regeneration and rooting than DPX cultivar. Findings of this study can be used for gene transfer and genetic engineering studies for Saman cultivar and other soybean cultivars.

Today, one of the ways to reduce production costs in poultry industrial units is to replace cost-effective grains such as wheat with corn in the poultry diet. However, the presence of non-starch polysaccharides in the cell wall of wheat has inevitably limited the uptake of nutrients by poultry. In this regard, one of the methods to remove non-starch polysaccharides is the use of xylanase enzyme additive in formulated diets based on wheat. For this purpose, the aim of this experiment was to clone the recombinant enzyme xylanase and investigate its effects on the performance of wheat-fed broiler chickens.

In current investigation, in the first step, xylanase gene, as a degradation enzyme of non-starch polysaccharides in wheat-based poultry diet, was cloned into plasmid pET-21a (+) in E. coli origami expression strain using heat shock method. IPTG inducer was used to induce gene expression and the expression result was observed on SDS-PAGE. Then, in the second step, the recombinant xylanase enzyme solution was added to the diet of one-hundred Ross 308 broilers in a completely randomized design. Then, the mean body weight gain (BWG), feed conversion ratio (FCR) and viscosity of jejunum contents were also measured.

The results of present study showed that the recombinant xylanase gene was transferred to origami and the cloning results of PCR confirmed the presence of a specific band of 966 bp. SDS-PAGE also showed a recombinant protein with a molecular weight of 35 kDa. Furthermore, the results of adding recombinant xylanase enzyme to wheat-based diet showed an increase in the weight of broilers and a decrease in feed conversion ratio and viscosity in the final period and the whole period compared to the control group at a probability level of 5%.

The results of the current research showed that the production of recombinant enzyme xylanase and its addition as a dietary supplement in wheat-based diets could increase the yield of broilers by breaking down arabinosylans in the wheat cell wall and the presence of xylanase enzyme in the diet is known as one of the important factors in enhancing the growth of broilers.

Maize (Zea mays L.) is a key cereal crop throughout the world and important model for plant genetics and biology. Establishment of an efficient transient gene expression system in maize facilitates plant functional genomics projects using gene silencing or heterologous protein overexpression strategies. The present study was aimed at optimizing an Agroinjection-mediated SCMV-based systemic heterologous gene expression system in maize.

Recombinant DNA encoding sugarcane mosaic virus (SCMV) containing the coding sequence of green fluorescent protein between the protein 1 (P1) and helper component‐proteinase (HC‐Pro) cistrons, in‐frame with the viral open reading frame, was introduced into the meristematic tissue, above the coleoptilar node, of maize seedlings via a direct Agroinoculation procedure. The efficiency of Agrobacterium tumefaciens strains EHA105 and GV3101 in delivering the recombinant vector into three and seven-day old seedlings of three maize varieties, including sweet corn (Iochief and Golden Bantam varieties) and dent corn (inbred line B73), was examined. Expression of GFP transgene in symptomatic Agroinoculated plants was assessed by confocal fluorescent microscopy and RT-PCR in comparison with controls.

Results of RT-PCR and confocal fluorescent microscopy revealed that: 1) A. tumefaciens GV3101 is significantly more successful in delivering the recombinant SCMV-based vector into maize plants than EHA105, 2) the percentage of GFP-expressing Golden Bantam plants is significantly higher than two other maize varieties, and 3) The effectiveness of growth stage of maize seedlings on the percentage of GFP expressing Agroinjected plants depends upon the interactions between Agrobacterium strain and maize genotype.

In conclusion, a combination of the Golden Bantam maize variety and A. tumefaciens strain GV3101 for direct Agroinjection of the SCMV-based vector into seedlings at the early two-leaf stage could be a fast and efficient system for investigating gene functions in maize.

Studies have shown that p32 is highly expressed in metabolically active and rapidly growing tissues, such as muscles, breast tumors, epidermis, and ovaries. One of the basic measures in domestic animals is the study of genes and proteins related to economic traits and their study at the cellular or chromosomal level. The aim of this study was to investigate the expression pattern of p32 gene in femur, humeral muscle, back muscle and back fat tissues of Kermani lambs.

Samples of femur, humeral muscle, back muscle and back fat tissues of Kermani lambs were collected (12 samples from 3 animals and 3 replicates from each tissue). After rapid freezing, the samples were stored in a -80 freezer. Total RNA was extracted using a standard kit. The quality and quantity of the extracted RNA were evaluated by agarose gel electrophoresis and using a nanodrop device. For cDNA synthesis, parstous cDNA synthesis kit was used. Real time PCR reaction by Syber Green method was used to evaluate the relative expression of genes. To analyze the data obtained from real time PCR, the Pfaffl method was used.

The quality of the extracted RNAs showed that the quality is appropriate and desirable. Observation of 18S and 28S bands in RNA indicated that it was intact and that no additional bands indicated its purity. Electrophoresis of PCR products on agarose gel and the results of real time PCR curves showed that the p32 gene was not expressed in femur, back muscle and back fat tissues of Kermani male lambs and was amplified only in the humeral muscle tissue of these animals. The observation of a single band in the range of 281 bp for the p32 gene in the humeral muscle tissue and the presence of a band in the range of 112 bp for the beta actin gene in all samples indicated the accuracy of the experiment and the amplification of the fragment.

Considering that in this study the expression of p32 gene in Kermani sheep at the age of 11 months was studied and the expression of this gene has been reported more in the embryonic period, so no expression of this gene was observed in femur, back muscle and back fat and its low expression in the humeral muscle tissue confirms the results of previous studies. Low expression of this gene in the humeral muscle tissue may indicate that the muscle is still growing at this age or that the tissue may be cancerous, as this gene is more expressed in tissues that are growing rapidly or are involved in cancer.