فصلنامه ژنتیک نوین
سال هفدهم شماره 4 (پیاپی 71، زمستان 1401)

In the last three decades, molecular farming has emerged as an alternative and cost-effective strategy for the successful production of hundreds of large-scale pharmaceutical recombinant proteins using plants. Because tumor cells need angiogenesis to grow and invade, therefore, targeting the most important angiogenic factor (vascular endothelial growth factor, VEGF) using the plant-based recombinant anti-VEGF can be an effective approach to inhibit angiogenesis and cancer treatment. The low expression level of recombinant protein is one of the major obstacles for the commercialization of molecular pharming. Therefore, numerous techniques have been developed to enhance protein expression, including codon optimization of protein sequences, appropriate subcellular targeting; the use of strong promoters, and the testing of different plant-based expression systems. In this paper, we investigate the effect of apoplast and endoplasmic reticulum (ER) targeting on Anti-VEGF recombinant nanobody accumulation in cucurbit plant. for this purpose, different molecular analyzes (RT-PCR, Dotblot, SDS-PAGE, Westernblot and ELISA) were performed and the results showed that ER targeting increased protein expression about 6-fold compared to its expression in the apoplast. Therefore, the ER is a more suitable site for expression of the target recombinant protein and other recombinant proteins than for apoplast.

The present study aimed to conduct a genome wide association studies (GWAS) based on Gene-set enrichment analysis for identifying the loci associated with growth traits using the 50K arrays. For this purpose, phenotypes records and genotypic data related to birth weight (BW), weaning weight (WW), six month weight (6MW) and 12 month weight (12MW) were obtained from 240 Hu sheep. Genome wide association study was performed with growth traits using TASSEL software. Using the biomaRt2 package R the SNP were assigned to genes if they were within the genomic sequence of the gene or within a flanking region of 15 kb up- and downstream of the gene. For the assignment of the genes to functional categories, the GO, KEGG, DAVID and PANTHER databases were used. By Gene set enrichment analysis, the biological pathways and candidate genes of regulation of skeletal muscle cell proliferation  and actin filament polymerization (MYOD1 and IGFBP4), regulation of anatomical structure morphogenesis, regulation of muscle system process and cell junction (BMP2, THBS1 and MYH10), regulation of skeletal muscle tissue development and regulation of anatomical structure size (FGF2, ANGPTL4, TGFBR3 and ACTR3), anatomical structure morphogenesis, positive regulation of ossification and cellular response to growth factor stimulus (MMP7, ACTA2, EGR1 and MYBPC3), for BW, WW, 6MW and 12MW were identified, respectively. The detected candidate genes played an important role in muscle structure development, glucose metabolism, Osteoclast differentiation, body size, skeletal muscle cell differentiation and regulation of ion calcium. These novel candidate genes identified in this study may be useful targets for clarifying our understanding of the molecular basis of growth traits in sheep. These results can provide new insight for future research into the genetic architecture of growth traits in sheep breeding program.

To evaluate the main and genotype × environment interaction effects of and to estimate and compare the correlation coefficients of genomic breeding values ​​of bread wheat bakery quality traits in an advanced bread wheat breeding population, an experiment in the form of alpha lattice design in three replications with 50 lines and checks in Chardaval research station Ilam Agricultural and Natural Resources Research and Education Center was conducted for two growing years in row. Analysis of variance simple and combined for bread wheat bread-making qualitative traits was performed. Combined analysis of variance indicated significant effects of genotype, environment and interactions of genotype × environment. In order to calculate genomic breeding values of ddRAD-Seq method and NextSeq TM 500 Illumina ® platform were used. The total number of correct SNPs recalled for 50% of the missing data were 3342, of which 1322, 1253 and 767were on the B, A & D genomes respectively. The highest SNPs on the 2B and 3B and the lowest on 4D chromosomes were identified. The correlation coefficients of genomic breeding values in the first, second and mean two years for crude protein, Falling number and Zeleny number traits was estimated by LASSO regression and elastic net regression and no model was fitted for SDS sediment volume.

Antifungal compounds of Trichoderma spp, including various lithic enzymes, have a important role in biological control of pathogenic agents. Xylan is a group of hemicellulose compounds which are present in the cell wall of plants and some algae. Green algae and green-blue algae having pectin, cellulose, xylan hemicellulose, arabinoxylan, etc., in their structures could be considered as suitable inducer and precursor of cellulase, xylanase, and pectinase enzymes production in fungi. Beta-xylanase genes, including Xyn1 and Xyn2 genes in Trichoderma species cause to produce the Glycoside hydrolase family 10, enzyme. In this study, the effect of aqueous extract of green microalgae Scenedesmus obliquus on enzyme production ability of four Trichoderma species was investigated. For this purpose, aqueous algae extract at concentrations of 500 and 1000 µg/ml were used during 4 and 8 days to enzyme production was investigated. Change in expression level of Xyln-related gene was analysied using real-timePCR method. The results of this study showed a positive and variable effect of algae extract in enzyme production in all the treated Trichoderma species. The maximum amount of enzyme secretion was observed at concentration of  1000 µg/ml during 8 days of treatment in T. reesei species. At this concentration and time period, T. atroviride showed the lowest amount of enzyme production. Gene expression analysis showed that, the maximum level of Xylan gene expression was occured in T. reesei species at concentration of 1000 µg/ml during 8 days of treatment. Based on these results, it can be concluded that the compounds in cellulosic structure of Scenedesmus mico-algae can have a considerable effect on enzyme secretion of fungi and then can be used as an enzyme stimulus in fungi in industrial and large -scale projects.

Cold stress is one of the sudden stresses and causes a lot of damage to agricultural products. Plants, despite having open cold stress tolerance mechanisms, are not able to act at the rate of stress development in the region. . The use of hormonal and quasi-hormonal compounds can reduce the damage caused by cold to some extent. For this purpose, the function of gamma-aminobutyric acid (GABA) on biochemical properties and expression of cold-tolerant genes in grape seedlings was investigated. In this study, to reduce cold stress losses from GABA leaf spray in the form of a randomized complete block design in three replications and in 3 concentrations (zero (control), 20.10 mM) on annual grape cuttings, Rish Baba cultivar (tolerant) and Yaghooti cultivar (susceptible) were used zero (control), 4, 8 and 10 hours) sampling was performed and in the first experiment biochemical parameters such as antioxidant content (total DPPH), phenol alcohol, and superoxide dismutase activity were examined, in the second experiment the treatment samples GABA at a concentration of 20 mM at 8 and 10 hours after application of stress in comparison with other treatments in both cultivars showed the most positive effect to counteract the effects of cold, so entered the molecular study and thus gene expression of CBF1, CBF2, CBF3 and CBF4 genes involved in cold were examined by Real Time-PCR. The results showed that GABA increases plant signaling and production of antioxidant compounds and is able to inhibit free radicals produced during stress. It also strengthens the signaling inside the plant and raises the expression level of resistance in grapes 8 and 10 hours after the application of cold stress. The transcription factor CBF2 has the highest expression level among GABA treated cultivars at a concentration of 20 mM has increased the resistance level in grape seedlings to an acceptable level.

Citrullus colocynthis is one of the most valuable medicinal plants belong to the Cucurbitaceae family and is a drought tolerant plant. This plant is an excellent source of various amino acids, including citrulline. Citrulline is an unnecessary alpha-amino acid, which is related with other amino acid such as Arginine and Ornithine. Citrulline is produced, from Ornithine and Carbamoyl Phosphate, in the urea cycle. Some genes such as Arginase, Argininosuccinate Lyase, and Nitric Oxide Synthase (NOS) involved in the synthesis of Citrulline. The aims of this study was to investigate the genes expression pattern that involved in the biosynthesis of Citrulline acid in different plant tissues of Citrullus colocynthis by qRT-PCR method. Different plant tissues of Citrullus colocynthis such as fruit, leaves, skin and flesh, immature and mature seeds were examined. Statistical analysis of the genes expression involved in Citrulline synthesis showed different gene expressions in different plant tissuese, so that in flesh and rind tissues of fruit were significantly higher than root, stem, leaf, immature and mature seed. In qRT-PCR analysis performed in different plant tissues of C. colocynthis, the expression of arginosuccinate lyase, arginosuccinate synthase and carbamyl phosphate synthase genes were the highest expression in skin and fleshy tissues. Also the arginase gene had the highest expression in rind, stem and leaf tissue, respectively. The expression of nitric oxide synthase gene in immature seeds was more expressed than rind tissue and was positively regulated, the lowest expression of NOS gene was in stem tissue. The relative expression of arginine succinate lyase gene in the present study was highest in immature seeds and lower in leaves than other tissues. The study of relative expression of arginine succinate synthase gene in different tissues of Citrullus colocynthis in the present study showed that stem and rind tissue have more expression. The highest relative expression of OTC enzyme gene was reported in Citrullus colocynthis fruit rind.

The enzyme Cheilanthifoline synthase (CFS), a member of the CYP719 protein family, is involved in the enzymatic conversion of reticulin to sanguinarine in plants. Sanguinarine is a Benzophenanthridine alkaloid that is widely found in plants of the family Papaveraceae, Famariaceae and Rotaceae. In the present study, the cDNA coding CFS was isolated from the roots of chelidonium majus. In the sequence analysis, the identification of the aromatic region, the binding region to hem group, and the EXXR conserved motif in the K helix confirmed the CFS enzyme belonging to the cytochrome P450 protein family. The expression pattern of CFS gene was evaluated in roots, stems and leaves of chelidonium majus treated with cerium dioxide and titanium dioxide nanoparticles at different times and concentrations. In plants treated with cerium dioxide, the highest expression of CFS gene was observed 72 hours after foliar spray of 100 mg/l in root and the lowest expression was observed in stem. In foliar spray of plants with titanium dioxide nanoparticles, 48 ​​hours after spray of 1000 mg/l, the highest expression increase of CFS gene showed in the root and the lowest expression was observed in the stem. The results of this study indicate an increase in transcripts of CFS gene in response to titanium dioxide and sodium dioxide nanoparticles in chelidonium majus; therefore, the obtained information from gene structure and the effectiveness of used elicitors can be useful in increasing the amount of sanguinarine alkaloid through metabolic engineering.

Genetic diversity identification is an important step for plant breeding procedure. Triticum urartu as the A-genome donor of wheat, is a valuable source for wheat breeding due to its rich allelic diversity for important traits such as agronomic characteristics, protein quality and biotic stress tolerance. In this study, the genetic diversity and population structure of 85 T.urartu accessions collected from different geographic regions of Iran were investigated using ISSR markers. 16 ISSR primers generated 164 fragments which all were polymorphic. The average of polymorphism information content (PIC) and marker index (MI) were 0.44 and 4.53 respectively, revealed a high resolving power of ISSR primers. The dendrogram generated using the NJ algorithm based on Jaccard’s dissimilarity coefficient, classified all 85 accessions into two main groups which was confirmed by Structure analysis. Analysis of molecular variance (AMOVA) showed a higher distribution of genetic diversity within populations (96%), than between them (4%) and according to the diversity indexes including number of effective alleles (Ne), Shannon’s index(I) and Nei’s gene diversity (He), there were an equal variation within seven investigated populations. The results showed a high amount of genetic variation among tested accessions, which provide further insights into conservation and future utilization of wild wheat resources. Besides, these results confirmed the efficiency of ISSR markers as reliable technique in estimating the genetic diversity and fingerprinting.

Genome editing based on CRISPR/Cas9 technology is rapidly expanding as a powerful tool for crop breeding. One of the advantages of this technology genome editing without introducing foreign DNA into plant genome.to achieve this, Cas9 and guide RNA need  delivered  to plant cell in form of protein-RNA complex (RNP). Hence the production of Cas9 enzyme in laboratory is the first step of CRISPR based on RNP method. Here, we evaluated the expression and purification of recombinant Cas9 protein in E. coli. In this study Cas9 gene was cloned into pBADM30 vector and transferred to the E. coli  CodonPlus strain. The result of colony PCR and double digest confirmed the presence of Cas9 gene into pBAD vector. Cas9 expression was induced by applying 0.2% L-Arabinose and then isolated from the total soluble protein. Cas9 protein was also purified from cell crude extract using Nickel affinity chromatography. The purification was analyzed by SDS-PAGE and show the presence of a 180 kDa band in the fraction collected during the elution step. The Cas9 protein in E. coli was successfully expressed and purified. According to the obtained results, it can be said that after measuring the catalytic activity of Cas9 enzyme, this enzyme can be used together with guide RNA and fabrication of RNP complex and its transfer to the plant by protoplast or electroporation method in further research.