Karim Imangholiloo; Nasrin Moshtaghi; Seyed Hasan Marashi; Abdolreza Bagheri; Ahmad Sharifi
Abstract
Cellulose which is extremely produced by plants, can be used for biofuel production but this function needs chemical or enzymatic digestion. Cellulose hydrolysis of plant wastes for ethanol production requires a mixture of three enzyme groups, including endoglucanases, exoglucanases, and beta-glucosidases. ...
Read More
Cellulose which is extremely produced by plants, can be used for biofuel production but this function needs chemical or enzymatic digestion. Cellulose hydrolysis of plant wastes for ethanol production requires a mixture of three enzyme groups, including endoglucanases, exoglucanases, and beta-glucosidases. The cellobiohydrolase enzyme (Cel6B) from Thermobifidia fusca has been used for cellulase activity extensively. This research aimed to express recombinant Cel6B enzyme in Pichia pastoris. For this purpose, cel6B gene in control of AOX1 promoter (methanol inducible) was introduced into Pichia pastoris. Amplification of cel6B gene was performed by PCR technique and then introduced into the Phil-S1 yeast vector. The recombinant construct contained the cel6B gene sequence and PHO1 signal sequence as secretion signal was transferred into Pichia pastoris GS115 strain. The transformed yeast cells expressed the recombinant Cel6B to yield 2.104 U (µmol/min)/ml of culture medium. Purified recombinant enzyme showed the best activity at 60 °C and pH 4.5 and this was agreed with optimum conditions for recombinant Cel6B enzymes which were produced in other systems. The purity of the enzyme was examined by SDS–PAGE technique, and a single band with a molecular weight about 59.6 kDa was observed. As cel6B gene sequence was not optimized for expression in the Pichia pastoris yeast, this could be one of the reasons for low level activity of recombinant Cel6B enzyme. This thermostable enzyme can be used for cellulolytic digestion of biomaterials in biofuel production research and other uses.
Azadeh Khadem; Nasrin Moshtaghi; Abdolreza Bagheri
Abstract
Somatic embryogenesis encompasses the same set of various developmental processes similar to zygotic embryogenesis. The conversion of somatic cells to embryos also requires stages of differentiation and reprogramming of cells. Since somatic embryogenesis is a complex process, a comprehensive investigation ...
Read More
Somatic embryogenesis encompasses the same set of various developmental processes similar to zygotic embryogenesis. The conversion of somatic cells to embryos also requires stages of differentiation and reprogramming of cells. Since somatic embryogenesis is a complex process, a comprehensive investigation is required to identify the effective gene networks and their interactions with environmental factors. As part of this study, bioinformatics tools and molecular studies were used to gain a better understanding of Arabidopsis thaliana somatic embryogenesis. The enriched pathways of somatic embryogenesis and their core-enriched genes were identified using gene set enrichment analysis. The results indicated that significant interaction between hormones helps to induce and develop somatic embryos. The gene ontology (including biological process, molecular function and cellular compartment) of core-enriched genes revealed that lipid storage and metabolism as well as stress response are the active biological pathways during somatic embryogenesis. In the protein-protein interaction network, TIR1/AFBs as auxin receptors exhibited the greatest number of interactions and proteins involved in lipid storage and metabolism acted as mediators between auxin receptors and ethylene perception. Also, Kyoto encyclopedia of genes and genomes analysis indicated that the metabolism of membrane lipids during somatic embryogenesis of Arabidopsis is primarily related to the biosynthesis of jasmonates and their derivatives. This process is initiated by Lypooxygenase proteins in the chloroplast, while Acyl-CoA oxidase 1 (ACX1) and Oxophytodienoate reductase 3 (OPR3) proceed this process in the peroxisome. The qRT-PCR analysis also confirmed the role of these genes during somatic embryogenesis, as the activity of these genes decreased at the beginning of 2,4-D treatment, but it increased during somatic embryogenesis. According to these results, jasmonates play an important role during somatic embryogenesis by mediating auxin signaling and stress response.
Mahdieh Yousefiara; Mohammad Ali Malboobi; Abdolreza Bagheri; Nasrin Moshtaghi
Abstract
Genetic engineering is a powerful technology of the present century that has revolutionized the agricultural, health, pharmaceutical and food industries worldwide. It is important to identify changes caused by transgenes that may be attributed to unintended traits in the risk assessment of genetically ...
Read More
Genetic engineering is a powerful technology of the present century that has revolutionized the agricultural, health, pharmaceutical and food industries worldwide. It is important to identify changes caused by transgenes that may be attributed to unintended traits in the risk assessment of genetically modified (GM) crops. Rhizomania, which is caused by beet necrotic yellow vein virus (BNYVV) infection, is considered to be a significant constraint in order to produce sugar beet worldwide. The resistance of transgenic sugar beet plants to the BNYVV was previously developed through RNA silencing by expression of hairpin RNA (hpRNA) structures. In the present study, the RNA sequencing (RNA-seq) analysis was performed in order to evaluate the transcriptional changes of an event of transgenic sugar beet plants, named 219-T3:S3-13.2 (S3), with the non-transgenic parental plants grown in virus-infected soil. The results of the present study indicate that there are only 0.9% differentially expressed genes (DEGs) at significant levels. The functional analysis shows alterations of transcription in lipids, amino acids, and carbohydrates metabolisms, cellular processes (autophagy), hormone signal transduction, and biosynthesis of secondary metabolites in the transgenic event, which are related to stress-adaption for which most of the genes were up-regulated. All in all, we conclude that the presence of the transgenes does not have substantial effects on the plant gene expression patterns. This work also indicates that RNA-seq analysis can be useful to evaluate the unintended effects and risk assessment of GM sugar beet plants.
Maria Beihaghi; Ahmad Reza Bahrami; Abdolreza Bagheri; Mohammad Zare Mehrjerdi
Abstract
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 ...
Read More
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.
Amir Ghaffar Shahriari; Abdolreza Bagheri; Mohammad Reza Bassami; Saeed Malekzadeh Shafaroudi; Ali Reza Afsharifar
Abstract
Newcastle is a significant avian disease continuing to cause considerable loss. Developments in genetic engineering have led to plant-based platforms for human and animal vaccine production. Recombinant vaccine production in hairy root systems have several advantages over stable expression in whole plants, ...
Read More
Newcastle is a significant avian disease continuing to cause considerable loss. Developments in genetic engineering have led to plant-based platforms for human and animal vaccine production. Recombinant vaccine production in hairy root systems have several advantages over stable expression in whole plants, including high growth rates, ready genetic manipulations, high levels of recombinant protein production, and the potential for bioreactor culture. In an attempt to develop a recombinant vaccine in hairy roots, the sequences encoding fusion (F) and haemagglutinin-neuraminidase (HN) epitopes of Newcastle disease virus were cloned in pBI121 expression vector which was then transferred into leaf disks of tobacco (Nicotiana tabaccum) 'Turkish' cultivar by means of Agrobacterium rhizogenes. Hairy roots developed on MS medium containing 50 mg/L kanamycin and 30 mg/L meropenem. Incorporation of the heterologous gene in the genome of hairy roots was confirmed by PCR. Expression analyses were performed by real-time PCR at transcription level and by dot-blot and ELISA assays at translation level, all confirming the expression of the heterologous gene and production of the recombinant protein.
Najme Javanmardi; Abdolreza Bagheri; Nasrin Moshtaghi; Ahmad Sharifi; Abbas Hemati Kakhki
Abstract
Saffron (Crocus sativus) is the most valuable and indigenous crop in Iran. The stigmas of flower are used as
a popular natural flavouring, colouring and medicinal agent. However, the market suffers from frauds in this
plant such as mixing with safflower petals due to high profit. Identification of ...
Read More
Saffron (Crocus sativus) is the most valuable and indigenous crop in Iran. The stigmas of flower are used as
a popular natural flavouring, colouring and medicinal agent. However, the market suffers from frauds in this
plant such as mixing with safflower petals due to high profit. Identification of these frauds with conventional
and biochemical methods is difficult and low sensitive. Therefore, application of molecular markers such as
random amplified polymorphic DNA (RAPD)/sequence characterized amplified regions (SCAR) is being
considered as an alternative. In this study, DNA was extracted from dry stigmas of 5 Saffron accessions and
dry petals of 7 safflower cultivars. RAPD reactions with ten 15-mer random primers resulted in two specific
monomorphic bands (500 and 700 bp) for safflower, while they were absent in saffron accessions. PCR
analysis with specific SCAR primers amplified two specific bands (414 and 589 bp) for safflowers in different
combinations of saffron stigmas and safflower petals. This was the case with very low rates or 1% of
safflower. Therefore, this method seems to be suitable for fraud identification of safflower petals in
commercial saffron samples.
Ahmad Reza Bahrami; Maria Beihaghi; Abdolreza Bagheri; Richard Leegood; Mehdi Ghabooli; Jafar Zolala; Farajollah Shahriari
Abstract
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 ...
Read More
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.
