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Abstract One of the causes of colon and gastric cancer is the dysregulation of carcinogenic genes, tumor inhibitors, and micro-RNA. The purpose of this study is to apply rs10811661 polymorphism in CDKN2A /B gene as an effective biomarker of colon cancer and early detection of gastric cancer. As a result,400 blood samples, inclusive of 200 samples from healthy individuals and 200 samples (100 samples from intestinal cancer,100 samples from stomach cancer) from the blood of someone with these cancers, to determine the genotype of genes in healthful and ill people through PCR-RFLP approach and Allelic and genotypic tests of SPSS software. To observe the connection between gastric cancer and bowel cancer risk and genotypes, the t-student test for quantitative variables and Pearson distribution for qualitative variables have been tested and the results have been evaluated using the Chi-square test. The effects confirmed that the highest frequency of TT genotypes is in affected individuals and CC genotype is in healthful individuals. In addition, it confirmed that women were more inclined than men to T3 tumor invasion and most grade II and III colon cancers, and in older sufferers with gastric cancer, the grade of tumor tended to be grade I. Among genetic variety and rs10811661, with invasiveness, there is a tumor size and degree in the affected person. In summary, our findings suggest that the rs10811661 polymorphism of the CDKN2A / B gene is strongly associated with the occurrence of intestinal cancer and stomach is linked to its potential role as a prognostic biomarker for the management of bowel cancer and stomach.

One of the newest diagnostic methods and treatment of cancer is to design new drugs. It is now possible to design a drug with desired properties in theory and evaluate its therapeutic effects through bioinformatics tools. Among the studied drugs, those based on cytokine genes, which increase the body's immunity against cancer, are of great interest. Cytokines are small proteins that play an essential role in cell signaling and can affect the function and behavior of surrounding cells. CCL21 chemokine is one of the cytokines that possess antitumor properties has the potential for chemoattraction of T lymphocytes and dendritic cells. Interleukin 1 beta (IL1β) is a cytokine involving different cellular activities such as the activation of neutrophils, B-Cells, and T-Cells. In the present study, we designed a drug-based cytokine gene to activate T cells and B cells by inserting defined CCL21 epitope and IL1β peptide sequences into a protein construct. Molecular dynamics simulation was performed in Linux space using Gromex software. Results of RMSD, RMSF, and the radius of gyration obtained from the simulation showed the stability of both proteins, which indicated that there are no significant conformational differences between the commercial CCL21 and recombinant form. The interaction of synthetic construct and human CCL21 with the CCR7 receptor was also investigated by HADDOCK software. Obtained results showed no differences between these proteins, and recombinant protein has the same structural and conformational characteristics as human commercial CCL21.

CCL21 has an essential role in anti-tumor immune activity. Epitopes of IL1β have adjuvant activity without causing inflammatory responses. CCR7 and its ligands play a vital role in the immune balance; specifically, in transport of T lymphocytes and antigen-presenting cells such as dendritic cells to the lymph nodes. This study aimed to produce epitopes of CCL21 and IL1β as a recombinant protein and characterize its in vitro anti-tumor and immunogenic activity. A codon-optimized ccl21/IL1β gene was designed and synthesized from human genes. Stability and binding affinity of CCL21/IL1β protein and CCR7 receptor were examined through in silico analyses. The construct was introduced into N. tabacum to produce this recombinant protein and the structure and function of CCL21/IL1β were examined. Purified protein from transgenic leaves generated a strong signal in SDS PAGE and western blotting assays. FTIR measurement and MALDI-TOF/TOF mass spectrography showed that ccl21/IL-1β was correctly expressed in tobacco plants. Potential activity of purified CCL21/IL1β in stimulating the proliferation and migration of MCF7 cancer cell line was investigated using the wound healing method. The results demonstrated a decrease in survival rate and metastasization of cancer cells in the presence of CCL21/IL1β, and IC50 of CCL21 on MCF7 cells was less than that of non-recombinant protein. Agarose assay on PBMCsCCR7+ showed that CCL21/IL1β has biological activity and there is a distinguishable difference between chemokinetic (CCL21) and chemotactic (FBS) movements. Overall, the results suggest that CCL21/IL1β could be considered an effective adjuvant in future in vivo and clinical tests.

IntroductionChemokine (C-C motif) ligand 21 (CCL21) has anti-tumor efficacy and used for immunotherapy based on cytokinins against cancer cells. Immune potentiating of CCL21 chemokine via DC and stromal cell-based approaches for effective recruitment and activation of APC and T cells for promotion of antitumor activity in lung cancer was evaluated. Plants are now gaining widespread acceptance as a general platform and as bioreactors for the large scale production of recombinant proteins. However, the technique suffers from major drawbacks such as low expression level and long time required for the production of the recombinant protein in plant tissues. The goal of the present study was to investigate the possibility of expressing the CCL21 protein in Cicer arietinum via agroinfiltration. In agroinfiltration, the suspension of Agrobacterium tumefaciens harboring the gene(s) of interest is infiltrated into plant leaves using a needle-free syringe. This technique has been carried out in a variety of plants with different experimental purposes and it was simple, cost effective and rapid procedure. Chickpea contain several minerals and phytochemicals components for human health, for example it has selenium, that plays essential roles in liver enzyme function, and helps detoxify some cancer-causing compounds in the body, Also selenium prevents inflammation and also decreases tumor growth rates. So cheakpea known as a valuable plant for the production of recombinant proteins chickpeas also contains folate, which plays a role in DNA synthesis and repair, thus preventing the formation of cancer cells from mutations in the DNA. Also it has saponins, which are phytochemicals that prevent cancer cells from multiplying and spreading throughout the body. High-fiber intakes from fruits and vegetables like chickpeas are associated with a lowered risk of colorectal cancer. Therefore, in this study, ccl21 gene of human was synthesized and transiently expressed in Cicer arietinum. Materials & MethodsTo examine the possibility of expressing the CCL21 protein in Cicer arietinum, a cDNA fragment encoding the ccl21 gene was synthesized de novo, modified with a Kozak sequence, a C-terminal hexa-histidine (6His) tag, and an endoplasmic retention signal (SEKDEL). The construct was subcloned into vector pBI121 and Agrobacterium colonies were verificated by PCR test. The resulting ccl21 plasmid was agro-infiltrated into Cicer arietinum. The relative gene expression of recombinant plant-produced ccl21 was measured by semi quantitatve RT-PCR and quantitative real-time PCR. Guided by the gene expression profile, CCL21 protein was extracted after 72 hours. A recombinant CCL21 protein was immunogenically detected by conjugated polyhistidine antibody in western blot, dot blot and ELISA assay. Results & Discussion The results of PCR assay confirmed presence of the synthetic construct in Agrobacterium clones. Also PCR test showed that, this construct was integrated in infiltrated leaves but the gene of interest is not integrated in the nuclear genome of plant cell. So, the transgene in plant tissue is higher than pure plasmid as control. RT- PCR and Real time PCR assay was also conducted to evaluate the expression of gene. Dot blot assay showed that, the protein sample obtained from transformed leaves generated a strong signal which is comparable to that of positive control, whereas wild type plant protein was not detectable. Also enzyme-linked immunosorbent assay (ELISA) and western blot showed that gene construt of ccl21 was expressed highly in transcription and translation level. Migration size of protein was detected at 15.5 KD by Western blotting. ELISA results showed that the CCL21 was expressed in the transfected leaves in high level. ConclusionThis paper is the first research about the transient expression of the chickpea-made CCL21 protein where a synthetic sequence was used for its expression. Here, the efficacy of agroinfiltration for expression of ccl21 gene in chickpea was illustrated. Agroinfiltration expedites the process of recombinant protein expression in plant tissues. Therefore, using agro-infiltration system in chickpea plant was the appropriate strategy for ccl21 gene production in the assayed plants. Therefore, this method makes it possible to evaluate efficacy of a potential recombinant vaccine in a short time.

Introduction: Plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues or organs under sterile conditions on a nutrient culture medium of known composition and widely used to produce clones of a plant in a method known as micropropagation. Plant research often involves growing new plants in a controlled environment. These may be plants that we have genetically altered in some way or may be plants of which we need many copies all exactly alike. These things can be accomplished through tissue culture of small tissue pieces from the plant of interest. These small pieces may come from a single mother plant or they may be the result of genetic transformation of single plant cells which are then encouraged to grow and to ultimately develop into a whole plant. Tissue culture techniques are often used for commercial production of plants as well as for plant research. Tobacco (Nicotiana tabacum L.) is one of the most important model plants used in the physiologic, genetic and tissue culture studies. The manipulation of tobacco genetic structure requires an efficient technique of gene transferring and regeneration. Whereas, the tobacco plant is a very effective bioreactor in the production of recombinant proteins, in this research we optimized the best tissue culture system and also, genetic transformation process of this plant. Materials and Methods: Our plant tissue culture protocols, Include helpful information for Murashige and Skoog media, plant growth regulators, plant growth hormones, plant transformation systems, and other products for plant tissue culture. For this purpose, different concentrations of sucrose and 4 combinations of growth regulators (BAP and NAA) on callus induction, direct shoot regeneration and rooting were examined in a factorial experiment based on completely randomized design with 3 replications. The sensitivity of tobacco explants to kanamycin was examined through the cultivation of them on the selective medium with different concentrations of antibiotic. For genetic transformation, agrobacterium tumifacious (GV3101) harboring plasmid pBI121 was used and the transgenic plants were confirmed by PCR analysis. Results and Discussion: The results of variance analysis and the means comparison showed that the best medium for callus induction was M1 (0.1 mg/l NAA and 1 mg/l BAP) with 15 g/l sucrose in the leaf explants, while the most direct shoot regeneration rate was obtained on the M1 medium with 30 g/l sucrose concentration. High-frequency of rooting was also influenced by 0/1 mg/l NAA and 60 g/l sucrose. So, supplementing the medium with NAA and BAP at different concentrations facilitated induction of multiple shoots from explants. NAA was proved to be the best and the number of shoots increased with increase in the concentration up to (0.1 mg/l), and exceeding this concentration resulted in decline in percent response as well as number of shoots was recorded shoot regeneration. The concentration of BAP was further increased a linear increase in the number of shoots was observed up to an optimal level (1 mg/l). Beyond the optimal concentration (1 mg/l), a decrease in the response as well as number of shoots was recorded due to profuse basal callusing. The effect of cytokinins on multiple shoot regeneration, higher concentrations of NAA found to be inhibitory for shoot regeneration because of huge callusing which hampered the growth and development of new shoots. Also different concentrations of sucrose have a different effect on the shoots and callus. The concentration of sucrose had significant effect on direct shoot regeneration. The main effect of sucrose concentration, concentration of 30 grams per liter, compared with a concentration of 15 grams per liter had the highest direct shoot regeneration. Concentration of 50 mg/l kanamycin could completely prevent the regeneration of untransformed explants so was used in the selective culture medium. Subsequently, the presence of nptII gene (798 bp) in the transgenic plants was confirmed and the transformation efficiency obtained by using the agrobacterium-mediated transformation was more than 95%. Conclusions In present research, an efficient in vitro regeneration protocol has been developed for tobacco, where different factors including the age of the explant and plant growth regulators were optimized for maximum propagation of tobacco. The results showed that regeneration and transformation method described here is highly efficient and fast for the introduction of any foreign gene directly in tobacco plant.

The main goal of this study was to investigate the possibility of expressing recombinant protein of C-C chemokine ligand 21 (CCL21) in Solanum lycopersicum via agroinfiltration. CCL21 is a chemokine can be used for anti-metastatic of cancer cell lines. To examine the expression of CCL21 protein in S. lycopersicum, the construct of ccl21 was synthesized. This construct was cloned into pBI121 and the resulting CCL21 plasmid was agro-infiltrated into S. lycopersicum leaves. Within three days after infiltration, Expression of the foreign gene was confirmed by quantitative Real-time PCR. A recombinant CCL21 protein was immunogenically detected by western blot, dot blot and ELISA assay. And results showed that the foreign gene was expressed in the transformed leaves in high level. Also scratch assay was used to investigate the role of this protein in anti-metastatic function. The results demonstrated anti-metastatic of cancer cells in the presence of this protein.

Studies on the genes contributing to the seed filling in chickpea and its protein content might be valuable in engineering plants with seeds of a higher nutritional value. A gene of interest is phosphoenolpyruvate carboxykinase (pepck), encoding a protein with a substantial role in the gluconeogenesis pathway. In the present study, the protein content (percentage) was measured in a number of cultivated chickpea genotypes, followed by comparison of the expression levels of pepck gene at different stages of seed filling in some of the genotypes. This study aims at revealing the relation between pepck transcript level with protein content of chickpea seeds which might, in the longer term, end at protein quality improvement of the crop through the gene manipulation procedures. The results which were verified by Real Time-PCR and Western blot techniques in four genotypes of plant, showed that, the amount of pepck expression was significantly higher at the stage of seed fillingthan at other stages in all of genotypes and the lowest levels of expression belonged to flowering and seed formation and the PEPCK protein level was higher in the high protein genotypes compared to the low protein genotypes.

Phosphoenolpyruvate Carboxykinase (PEPCK) has been shown to be present in plants andanimals, playing different metabolic roles in different tissues. The basic role of this enzyme is itscontribution in the gluconeogenesis pathway. Evidences have shown that PEPCK may play a role inmetabolism of nitrogenous compounds in developing seeds of legumes. In this research, pepck geneexpression and the occurrence of PEPCK protein and its activity in different genotypes of chickpea(Cicer arietinume L.) were determined. Two low protein genotypes (MCC291 & MCC373) and twohigh protein genotypes (MCC458 & MCC053) out of 20 chickpea genotypes were selected, fromwhich the total RNA was extracted through different stages of seed development. The expression ofchickpea pepck gene was estimated by semi-quantitative RT-PCR. The results of RT-PCR showed thattwo isoforms of this gene were expressed in high protein genotypes, whereas in the low proteingenotypes were not expressed. The differential expression of pepck gene is perhaps related to thepossible role of Phosphoenolpyrovate Carboxykinase in protein content of chickpea seeds.Key words: Chickpea, Nitrogen metabolism, Phosphoenolpyruvate Carboxykinase, Seed development

Abstract Phosphoenolpyruvate Carboxykinase, encoded by the pepck gene, plays an important role in gluconeogenesis. It also seems to be important in metabolism of nitrogenous compounds in developing seeds of legumes, including amides and ureides which are then transformed into amino acids, necessary for the synthesis of storage proteins. In this research, pepck gene expression in mRNA level, in different genotypes of chickpea (Cicer arietinum L.), was determined. Two low protein genotypes (MCC291 and MCC373) and two high protein genotypes (MCC458 and MCC053) out of 20 chickpea genotypes were selected. Total RNA were extracted through different stages of seed development, and the expression of the pepck gene was estimated by semi-quantitative RT-PCR. The results of the RT-PCR showed that two isoforms of this gene are expressed in high protein genotypes, whereas in the low protein genotypes, the expression of these isoforms was not obvious. Also this method showed a differential expression of pepck gene in different stages of flowering and seed development. pepck gene is expressed in higher levels during the sheet formation and developing seeds compared to the flowering and seed formation stages. Probably, the differential expression of pepck gene is related to its possible role in metabolism of seed components, particularly in determination of the protein content of chickpea seeds.