One year old olive (Olea europaea L. cv. Zard) plants were treated with 0.5, 1, and 2 mM salicyli... more One year old olive (Olea europaea L. cv. Zard) plants were treated with 0.5, 1, and 2 mM salicylic acid (SA) and then exposed to nonfreezing and freezing temperatures (-5,-10, and-20°C) for 10 h. Untreated plants served as a control. Exposure to freezing temperatures caused a considerable increase in ion leakage and lipid peroxidation in olive leaves. Treatment with suitable exogenous SA (1.0 mM) prevented the increase in the ion leakage and lipid peroxidation caused by freezing temperatures, especially at-5 and-10°C. SA induced freezing tolerance was accompanied by increased activities of antioxidant enzymes, such as gua iacol peroxidase, catalase, ascorbate peroxidase, and polyphenol oxidase, as compared to control plants. Pro line, total phenolic content, and antioxidant capacity of olive leaves were declined significantly after exposure to freezing temperature, and their content decreased with lowering of freezing temperatures, while treatment with 1 mM SA induced a significant increase in their content. As a summary of these results, suitable concen tration of SA (1 mM) could enhance freezing tolerance of olive plant by increasing antioxidant enzyme activ ities and decreasing MDA content through cell membrane integrity maintenance.
By using amenable MS based medium containing 4 mg l-1 2,4-Dichlorophenoxyacetic acid (2,4-D), 0.4... more By using amenable MS based medium containing 4 mg l-1 2,4-Dichlorophenoxyacetic acid (2,4-D), 0.4 mg l-1 benzylaminopurine (BAP), 30 g l-1 sucrose, 8 g l-1 Agar-agar, qualitative and quantitative traits of calluses initiated from four genetically and commercially valuable Northern Iranian rice cultivars including Hashemi, Hasani, Gerdeh, and Gharib were studied. Five seeds were placed in each Petri dish and three replicates of eight Petri dishes per replicate were incubated in a growth chamber at 25 ±2 °C in the dark and the averages for every replicate were employed in the analyses. Several important parameters related to callogenesis of the cultivars including rate of non-viable seeds, necrotic, scutellar, slow growing, and non-scutellar calluses, simultaneous callus induction from scutellar and non-scutellar tissues, seeds with appropriate callus, and root emergence were compared. Accordingly, calli of Gharib and Hashemi were highly responsive in callogenesis, while Gerdeh and Hasani produced dissatisfying calluses. Necrotic calluses, scutellar calli, and non-viable seeds were positively correlated with each other; although they were negatively correlated with non-scutellar calli, simultaneous scutellar and non-scutellar calli induction, and root emergence. The results of the present study are expected to be the first promising step to generate genetically manipulated Iranian indigenous rice cultivars.
Iranian Journal of Genetics and Plant Breeding, Sep 14, 2021
Agrobacterium tumefaciens-based plant transformation is a natural and ideal way of introducing ge... more Agrobacterium tumefaciens-based plant transformation is a natural and ideal way of introducing genes into plant genomes. The system is based primarily on tissue culture techniques, including cell differentiation and plant regeneration, which might produce somatic cell mutations. In the present research, wheat plants were transformed by a non-tissue culture approach. Accordingly, mature embryos were inoculated with A. tumefaciens at an early stage of germination. A variety of different treatments, such as Agrobacterium strains (EHA105 and LBA4404 harboring pCAMBIAl105.1R), levels of glucose, concentrations of acetosyringone (0, 50, 100 and 200 µM), and types of wheat cultivars (Azar2, Alvand, and Sardari) were studied. The transformation efficiency was determined by using the number of resistant leaves to hygromycin, histochemical GUS analysis of leaf tissues, as well as PCR analysis of three transgenes located within the T-DNA region. The results show maximum efficiency obtained when 200 µM acetosyringone in combination with 15% glucose are used in the induction medium. In addition, the relative expression analysis of virulence genes by qRT-PCR revealed that VirB2 and VirD2 are significantly up-regulated when 200 µM acetosyringone and 15% glucose are used as inducers and carbon sources. Therefore, Vir genes inducing factors as well as three-step culture of Agrobacterium should be considered as major components of any wheat transformation protocol. The Putative transgenic T1 seeds were soaked and germinated in hygromycin solution, which the seedlings further analyzed with aforementioned methods. Based on the results, a modified Agrobacterium-mediated transformation protocol is presented. The current results suggest an inexpensive, quick, and efficient approach to transform wheat genome.
Background: Histone deacetylation plays an essential role in transcriptional regulation of cell c... more Background: Histone deacetylation plays an essential role in transcriptional regulation of cell cycle progression and other evolutionary processes. Several results confirm the importance of the latest found HDAC11 gene to deacetylate histone core in neurons and their supportive cells in developing the vertebrate Central Nervous System (CNS). Objectives: This study investigates the HDAC11 potential role in early chicken CNS development by studying its mRNA expression profile which may have unique means in studying human subjects. Materials & Methods: Chicken HDAC11 RNAs were reverse transcribed to cDNAs, and the amount of chHDAC11 transcripts was measured by ΔCT mean calculation using the real-time quantitative PCR method. One-way ANOVA and Duncan's analysis (SigmaStat software version 4.0) were used to test the statistical significance of the results. The levels of significance were set at P≤0.05. Quantitative data are presented as Mean±SD. Results: The amount of HDAC11 mRNAs gradually increases, at least 2-3 times, from as early as day 14 (E14/HH40) of prenatal cortex formation to day P0 (E20=HH45) and continue to increase to day 40 in both cortical and hippocampal regions of the postnatal chicken brain during development (*P≤0.05). HDAC11 mRNA is not only expressed in the postnatal cortex and hippocampi regions but also-for the first time-in the developing brain during the prenatal period. Conclusion: Our results show a possibly important role for the latest found HDAC11 conserved gene in the development of vertebrates' embryonic brain, which in turn may have a significant impact on understanding human brain development.
International Journal of Agriculture Innovations and Research, 2014
Gramineous crop plants including important cereals like rice (Oryza sativa L.) were classified as... more Gramineous crop plants including important cereals like rice (Oryza sativa L.) were classified as recalcitrant to transformation because of their both low morphogenic potential and transformation efficiency. An efficient in planta transformation method, via physical wounding of the embryo region was conducted in two rice landraces (O. sativa ssp. Indica) where the seeds were soaked in water for 48 hours and pierced with an Agrobacteriumcoated needle. Two strains of A. tumefaciens (EHA105 and LBA4404) harbouring pCAMBIA1105.1r vector were used. A three step method in cell culture was applied for vir gene induction in Agrobacterium. Integration and presence of the transgene into the genome of putative transgenic rice plants was confirmed using polymerase chain reaction, resistance of leaf tissues to hygromycin and histiochemical GUS assay. In a comparative study, EHA105 strain in the presence of 100 µM acetosyringone on 'Hashemi' cultivar had a significantly higher transformation efficiency (~9.0%) compared to the other treatments. Ninety T 0 seedlings were selected randomly and analysed further at T 1 generation, which ~28% confirmed to be positive.
Glycyrrhiza glabra is one of the most important and well-known medicinal plants which produces va... more Glycyrrhiza glabra is one of the most important and well-known medicinal plants which produces various triterpene saponins such as glycyrrhizin. Beta-amyrin 11-oxidase (CYP88D6) plays a key role in engineering pathway of glycyrrhizin production and converts an intermediated beta-amyrin compound to glycyrrhizin. In this study, pBI121 GUS-9 :CYP88D6 construct was transferred to G. glabra using Agrobacterium rhizogene ATCC 15834. The quantitation of transgene was measured in putative transgenic hairy roots using qRT-PCR. The amount of glycyrrhizin production was measured by HPLC in transgenic hairy root lines. Gene expression analysis demonstrated that CYP88D6 was over-expressed only in one of transgenic hairy root lines and was reduced in two others. Beta-amyrin 24-hydroxylase (CYP93E6) was significantly expressed in one of the control hairy root lines. The amount of glycyrrhizin metabolite in over-expressed line was more than or similar to that of control hairy root lines. According to the obtained results, it would be recommended that multi-genes of glycyrrhizin biosynthetic pathway be transferred simultaneously to the hairy root in order to increase glycyrrhizin content.
Licorice is a well-known medicinal plant, containing various secondary metabolites of triterpenoi... more Licorice is a well-known medicinal plant, containing various secondary metabolites of triterpenoid and phenolic families. The aim of this study is to evaluate the effect of salinity stress on the expression of key genes involved in the biosynthetic pathway of triterpenoids such as glycyrrhizin, betulinic acid, soyasaponins, and phytosterols in licorice root, as well as providing a phonemic platform to characterize antioxidant properties, glycyrrhizin, and total phenolic content. This study also includes measuring the gene expression level and glycyrrhizin content in leaves and roots of control plants. The studied genes included squalene synthase (SQS1 and SQS2), β-amyrin synthase (bAS), lupeol synthase (LUS), cycloartenol synthase (CAS), β-amyrin 11-oxidase (CYP88D6), and β-amyrin 24-hydroxylase (CYP93E6). Our results revealed that all of the mentioned genes were upregulated following the stress condition with different transcription rates. The highest increase (12-fold) was observed for the expression of the LUS gene, which is related to the betulinic acid pathway. Also, the highest content of glycyrrhizin was observed at 72 h posttreatment, which was consistent with the upregulated transcription levels of the glycyrrhizin pathway genes especially SQS1 and CYP88D6 at the same time. Correlation and stepwise regression analysis proved the key role of SQS1 gene in the biosynthetic pathway of glycyrrhizin. Antioxidant activity and phenolic content also were increased following stress condition. A comparison between the expression levels of SQS1 and other genes involved in the production of glycyrrhizin, phytosterols, and soyasaponins revealed a similar transcription trend, which shows the gene expression in the roots was significantly higher than the leaves. In contrast, SQS2 and LUS genes displayed a higher expression in leaf tissues. The genes related to betulinic acid biosynthetic pathway exhibited an expression rate different from other triterpenoid pathway genes, which could be observed in the leaves and roots of control plants and the roots of salt-treated plants. Furthermore, results showed that these two SQS genes have different expression rates due to different plant tissues (roots and leaves) and stress conditions. Importantly, in contrast to previous reports, we detected the glycyrrhizin in leaf tissues. This result may indicate the presence of a different genetic background in native Iranian licorice germplasm. Keywords Gene expression. Glycyrrhiza glabra. Glycyrrhizin. Quantitative real-time PCR. Secondary metabolites Abbreviations bAS β-Amyrin synthase CAS Cycloartenol synthase CYP88D6 β-Amyrin 11-oxidase CYP93E6 β-Amyrin 24-hydroxylase FDP Farnesyl diphosphate GA3 Gibberellic acid HPLC High-performance liquid chromatography LUS Lupeol synthase MeJA Methyl jasmonate OSCs Oxidosqualene cyclases QRT-PCR Quantitative reverse transcription PCR SQS Squalene synthase
Protein domains with similarity to plant strictosidine synthase-like (SSL) sequences have been un... more Protein domains with similarity to plant strictosidine synthase-like (SSL) sequences have been uncovered in the genomes of all multicellular organisms sequenced so far and are known to play a role in animal immune responses. Among several distinct groups of Arabidopsis thaliana SSL sequences, four genes (AtSSL4-AtSSL7) arranged in tandem on chromosome 3 show more similarity to SSL genes from Drosophila melanogaster and Caenorhabditis elegans than to other Arabidopsis SSL genes. To examine whether any of the four AtSSL genes are immune-inducible, we analysed the expression of each of the four AtSSL genes after exposure to microbial pathogens, wounding and plant defence elicitors using real-time quantitative RT-PCR, Northern blot hybridisation and Western blot analysis with antibodies raised against recombinant AtSSL proteins. While the AtSSL4 gene was constitutively expressed and not significantly induced by any treatment, the other three AtSSL genes were induced to various degrees by plant defence signalling compounds, such as salicylic acid, methyl jasmonate and ethylene, as well as by wounding and exposure to the plant pathogens Alternaria brassicicola and cucumber mosaic virus. Our data demonstrate that the four SSL-coding genes are regulated individually, suggesting specific roles in basal (SSL4) and inducible (SSL5-7) plant defence mechanisms.
OBJECTIVE: HDACs are among transcriptional regulatory elements that regulate key features of prol... more OBJECTIVE: HDACs are among transcriptional regulatory elements that regulate key features of proliferation and differentiation in all cell types including cancerous. They may also interfere in such stages of cancer development as migration, invasion, multi-drug resistance and angiogenesis. Proven information about HDAC1 role in development of bladder cancer is limited only to cell lines in vitro. The lack of a comprehensive clinical in vivo study led us to evaluate HDAC1 expression in human clinical specimens. METHODS: We analyzed a large group of bladder cancer patients. The presence of hHDAC1 mRNAs were tracked using specifi c HDAC1 primers in cancer samples and the quantity of HDAC1 transcripts were quantifi ed using real time qPCR method and was compared to those of normal bladder samples from healthy patients. RESULTS: HDAC1 mRNA expression was signifi cantly elevated in Bladder cancer specimens. To our knowledge, this result is the fi rst, showing an elevation in vivo in HDAC1 mRNA levels in clinically cancerous tissue of patients with bladder cancer. CONCLUSIONS: We conclude that hHDAC1 overexpression might be implicated in bladder cancer tumorigenesis and that the over-expressed HDAC1 mRNA might be a potential diagnostic marker and, a target for treatment of bladder cancer using HDACi-drugs in future (Tab. 2, Fig. 2, Ref. 30).
Strictosidine synthase is a key enzyme in the biosynthesis of plant alkaloids. The homologous of ... more Strictosidine synthase is a key enzyme in the biosynthesis of plant alkaloids. The homologous of the strictosidine synthase domain has been found in different organisms such as Arabidopsis thaliana. Bioinformatics and expression studies on one of the strictosidine synthase-like gene (SSL6) in Arabidopsis plants showed that gene responded to the signaling molecules, fungal and viral pathogens. The functional role of SSL6 has been further studied using an Arabidopsis knockout mutant with a T-DNA inserted into exon 1. The plants were inoculated with Alternaria brassicicola, which had incompatible interaction with A. thaliana. The fungal biomass was quantified by quantitative real-time PCR and spore count techniques. The assays indicated that T-DNA mediated ssl6 mutant was more susceptible than Col-0 genotype. The chlorophyll content and the size of necrotic lesions were significantly smaller in the ssl6 mutant compared with the Col-0. This is the first report with reference to the functional analysis of SSL6 suggesting that might be SSL6 plays an important role, among other factors, in plant defense against A. brassicicola.
Genetic Engineering and Biosafety Journal, Oct 10, 2012
enescence is a final developmental stage of leaf, which is very important as genetic and physiolo... more enescence is a final developmental stage of leaf, which is very important as genetic and physiological aspects. Many genes are activated at this stage and most of them show remarkable transcript. The function of senescence is to control regulatory physiological changes. This include cessation of photosynthesis, chloroplast degradation, chlorophyll loss and protein breakdown. Senescence can be initiated by a wide variety of different internal and external factors, as well as being an essential part of plant development; senescence in leaves is also induced prematurely by a number of different environmental stresses. Since plants cannot escape from adverse environmental conditions senescence is one mechanism that plants have evolved to cope with such problems. Interestingly, senescence could be induced in plants even after harvest. This phenomenon is observed in such vegetables like broccoli, lettuce and cabbage. Many different senescence-enhanced genes have been isolated, characterized and cloned. Expression analysis of these genes showed a broad range of expression some time before phenotypic changes to last stage of senescence. So it seems that many signaling pathways should be involved in this process.
Licorice is the roots and stolons of Glycyrrhiza uralensis which have several chemical compounds.... more Licorice is the roots and stolons of Glycyrrhiza uralensis which have several chemical compounds. Triterpene saponins such as glycyrrhizin and glycyrrhetinic acid and flavonoids like liquiritin, isoliquiritigenin and glabron are main compounds detected in liquorice root. The plant's major constituent is a glycyrrhizin. The β-amyrin 11-oxidase catalyzes the sequential two-step oxidation of β-amyrin in C-11 to produce 11-oxo-β-amyrin, a possible biosynthetic intermediate between β-amyrin and glycyrrhizin. In this study, the total RNA was extracted from licorice roots and cDNA synthesis, then PCR products were cloned into pTZ57R/T vector. Sequencing confirmed piece length of 1482 bp that encodes a protein of 493 amino acid residues. The results of alignment showe d 99% similarity to βamyrin sequence of Glycyrrhiza uralensis. Subcellular studies using Softberry and Psort software showed that the activity of this protein is in endoplasmic reticulum. Moreover the protein has a signal peptide and is targeted to the secretory pathway. The results of phylogenetic tree determined most similar amino acid sequence to the CYP88D subfamily of cytochrome P450. These findings can be used for nucleotide or protein manipulation and transformation.
The entomopathogenic fungi-like Beauveria bassiana must penetrate via the integument of an insect... more The entomopathogenic fungi-like Beauveria bassiana must penetrate via the integument of an insect to reach the hemocoel. Since proteins are the molecules responsible for integument strength in insects, the proteins must synthesise the cuticle degrading proteases which will then enable the proteases to penetrate. It is important to determine the biochemical properties of these proteases so that fungal virulence can be better understood. In the current study, a recently collected isolate of B. bassiana, namely AM-118, was inoculated in liquid media containing 0.5% of Andrallus spinidens Fabricus cuticle to obtain specific proteases. The crude samples were purified via a three step process using ammonium sulfate, Sepharyl G-100, and DEAE-Cellulose Fast Flow. The results revealed two proteases known as subtilisin-like (Pr1), and trypsin-like (Pr2), with the molecular weights of 105 and 103 kDa. The optimal pH and temperature values were found to be 8 and 35°C for Pr1 and 8 and 40°C for Pr2, respectively. Inhibitors like AEBSF, EDTA, TPCK, and phenanthroline significantly affected proteolytic activities. Here, we reported two fungal proteases by high molecular weight from an Iranian isolate of B. bassiana. These findings will help us to better understand fungal virulence against insects.
One year old olive (Olea europaea L. cv. Zard) plants were treated with 0.5, 1, and 2 mM salicyli... more One year old olive (Olea europaea L. cv. Zard) plants were treated with 0.5, 1, and 2 mM salicylic acid (SA) and then exposed to nonfreezing and freezing temperatures (-5,-10, and-20°C) for 10 h. Untreated plants served as a control. Exposure to freezing temperatures caused a considerable increase in ion leakage and lipid peroxidation in olive leaves. Treatment with suitable exogenous SA (1.0 mM) prevented the increase in the ion leakage and lipid peroxidation caused by freezing temperatures, especially at-5 and-10°C. SA induced freezing tolerance was accompanied by increased activities of antioxidant enzymes, such as gua iacol peroxidase, catalase, ascorbate peroxidase, and polyphenol oxidase, as compared to control plants. Pro line, total phenolic content, and antioxidant capacity of olive leaves were declined significantly after exposure to freezing temperature, and their content decreased with lowering of freezing temperatures, while treatment with 1 mM SA induced a significant increase in their content. As a summary of these results, suitable concen tration of SA (1 mM) could enhance freezing tolerance of olive plant by increasing antioxidant enzyme activ ities and decreasing MDA content through cell membrane integrity maintenance.
By using amenable MS based medium containing 4 mg l-1 2,4-Dichlorophenoxyacetic acid (2,4-D), 0.4... more By using amenable MS based medium containing 4 mg l-1 2,4-Dichlorophenoxyacetic acid (2,4-D), 0.4 mg l-1 benzylaminopurine (BAP), 30 g l-1 sucrose, 8 g l-1 Agar-agar, qualitative and quantitative traits of calluses initiated from four genetically and commercially valuable Northern Iranian rice cultivars including Hashemi, Hasani, Gerdeh, and Gharib were studied. Five seeds were placed in each Petri dish and three replicates of eight Petri dishes per replicate were incubated in a growth chamber at 25 ±2 °C in the dark and the averages for every replicate were employed in the analyses. Several important parameters related to callogenesis of the cultivars including rate of non-viable seeds, necrotic, scutellar, slow growing, and non-scutellar calluses, simultaneous callus induction from scutellar and non-scutellar tissues, seeds with appropriate callus, and root emergence were compared. Accordingly, calli of Gharib and Hashemi were highly responsive in callogenesis, while Gerdeh and Hasani produced dissatisfying calluses. Necrotic calluses, scutellar calli, and non-viable seeds were positively correlated with each other; although they were negatively correlated with non-scutellar calli, simultaneous scutellar and non-scutellar calli induction, and root emergence. The results of the present study are expected to be the first promising step to generate genetically manipulated Iranian indigenous rice cultivars.
Iranian Journal of Genetics and Plant Breeding, Sep 14, 2021
Agrobacterium tumefaciens-based plant transformation is a natural and ideal way of introducing ge... more Agrobacterium tumefaciens-based plant transformation is a natural and ideal way of introducing genes into plant genomes. The system is based primarily on tissue culture techniques, including cell differentiation and plant regeneration, which might produce somatic cell mutations. In the present research, wheat plants were transformed by a non-tissue culture approach. Accordingly, mature embryos were inoculated with A. tumefaciens at an early stage of germination. A variety of different treatments, such as Agrobacterium strains (EHA105 and LBA4404 harboring pCAMBIAl105.1R), levels of glucose, concentrations of acetosyringone (0, 50, 100 and 200 µM), and types of wheat cultivars (Azar2, Alvand, and Sardari) were studied. The transformation efficiency was determined by using the number of resistant leaves to hygromycin, histochemical GUS analysis of leaf tissues, as well as PCR analysis of three transgenes located within the T-DNA region. The results show maximum efficiency obtained when 200 µM acetosyringone in combination with 15% glucose are used in the induction medium. In addition, the relative expression analysis of virulence genes by qRT-PCR revealed that VirB2 and VirD2 are significantly up-regulated when 200 µM acetosyringone and 15% glucose are used as inducers and carbon sources. Therefore, Vir genes inducing factors as well as three-step culture of Agrobacterium should be considered as major components of any wheat transformation protocol. The Putative transgenic T1 seeds were soaked and germinated in hygromycin solution, which the seedlings further analyzed with aforementioned methods. Based on the results, a modified Agrobacterium-mediated transformation protocol is presented. The current results suggest an inexpensive, quick, and efficient approach to transform wheat genome.
Background: Histone deacetylation plays an essential role in transcriptional regulation of cell c... more Background: Histone deacetylation plays an essential role in transcriptional regulation of cell cycle progression and other evolutionary processes. Several results confirm the importance of the latest found HDAC11 gene to deacetylate histone core in neurons and their supportive cells in developing the vertebrate Central Nervous System (CNS). Objectives: This study investigates the HDAC11 potential role in early chicken CNS development by studying its mRNA expression profile which may have unique means in studying human subjects. Materials & Methods: Chicken HDAC11 RNAs were reverse transcribed to cDNAs, and the amount of chHDAC11 transcripts was measured by ΔCT mean calculation using the real-time quantitative PCR method. One-way ANOVA and Duncan's analysis (SigmaStat software version 4.0) were used to test the statistical significance of the results. The levels of significance were set at P≤0.05. Quantitative data are presented as Mean±SD. Results: The amount of HDAC11 mRNAs gradually increases, at least 2-3 times, from as early as day 14 (E14/HH40) of prenatal cortex formation to day P0 (E20=HH45) and continue to increase to day 40 in both cortical and hippocampal regions of the postnatal chicken brain during development (*P≤0.05). HDAC11 mRNA is not only expressed in the postnatal cortex and hippocampi regions but also-for the first time-in the developing brain during the prenatal period. Conclusion: Our results show a possibly important role for the latest found HDAC11 conserved gene in the development of vertebrates' embryonic brain, which in turn may have a significant impact on understanding human brain development.
International Journal of Agriculture Innovations and Research, 2014
Gramineous crop plants including important cereals like rice (Oryza sativa L.) were classified as... more Gramineous crop plants including important cereals like rice (Oryza sativa L.) were classified as recalcitrant to transformation because of their both low morphogenic potential and transformation efficiency. An efficient in planta transformation method, via physical wounding of the embryo region was conducted in two rice landraces (O. sativa ssp. Indica) where the seeds were soaked in water for 48 hours and pierced with an Agrobacteriumcoated needle. Two strains of A. tumefaciens (EHA105 and LBA4404) harbouring pCAMBIA1105.1r vector were used. A three step method in cell culture was applied for vir gene induction in Agrobacterium. Integration and presence of the transgene into the genome of putative transgenic rice plants was confirmed using polymerase chain reaction, resistance of leaf tissues to hygromycin and histiochemical GUS assay. In a comparative study, EHA105 strain in the presence of 100 µM acetosyringone on 'Hashemi' cultivar had a significantly higher transformation efficiency (~9.0%) compared to the other treatments. Ninety T 0 seedlings were selected randomly and analysed further at T 1 generation, which ~28% confirmed to be positive.
Glycyrrhiza glabra is one of the most important and well-known medicinal plants which produces va... more Glycyrrhiza glabra is one of the most important and well-known medicinal plants which produces various triterpene saponins such as glycyrrhizin. Beta-amyrin 11-oxidase (CYP88D6) plays a key role in engineering pathway of glycyrrhizin production and converts an intermediated beta-amyrin compound to glycyrrhizin. In this study, pBI121 GUS-9 :CYP88D6 construct was transferred to G. glabra using Agrobacterium rhizogene ATCC 15834. The quantitation of transgene was measured in putative transgenic hairy roots using qRT-PCR. The amount of glycyrrhizin production was measured by HPLC in transgenic hairy root lines. Gene expression analysis demonstrated that CYP88D6 was over-expressed only in one of transgenic hairy root lines and was reduced in two others. Beta-amyrin 24-hydroxylase (CYP93E6) was significantly expressed in one of the control hairy root lines. The amount of glycyrrhizin metabolite in over-expressed line was more than or similar to that of control hairy root lines. According to the obtained results, it would be recommended that multi-genes of glycyrrhizin biosynthetic pathway be transferred simultaneously to the hairy root in order to increase glycyrrhizin content.
Licorice is a well-known medicinal plant, containing various secondary metabolites of triterpenoi... more Licorice is a well-known medicinal plant, containing various secondary metabolites of triterpenoid and phenolic families. The aim of this study is to evaluate the effect of salinity stress on the expression of key genes involved in the biosynthetic pathway of triterpenoids such as glycyrrhizin, betulinic acid, soyasaponins, and phytosterols in licorice root, as well as providing a phonemic platform to characterize antioxidant properties, glycyrrhizin, and total phenolic content. This study also includes measuring the gene expression level and glycyrrhizin content in leaves and roots of control plants. The studied genes included squalene synthase (SQS1 and SQS2), β-amyrin synthase (bAS), lupeol synthase (LUS), cycloartenol synthase (CAS), β-amyrin 11-oxidase (CYP88D6), and β-amyrin 24-hydroxylase (CYP93E6). Our results revealed that all of the mentioned genes were upregulated following the stress condition with different transcription rates. The highest increase (12-fold) was observed for the expression of the LUS gene, which is related to the betulinic acid pathway. Also, the highest content of glycyrrhizin was observed at 72 h posttreatment, which was consistent with the upregulated transcription levels of the glycyrrhizin pathway genes especially SQS1 and CYP88D6 at the same time. Correlation and stepwise regression analysis proved the key role of SQS1 gene in the biosynthetic pathway of glycyrrhizin. Antioxidant activity and phenolic content also were increased following stress condition. A comparison between the expression levels of SQS1 and other genes involved in the production of glycyrrhizin, phytosterols, and soyasaponins revealed a similar transcription trend, which shows the gene expression in the roots was significantly higher than the leaves. In contrast, SQS2 and LUS genes displayed a higher expression in leaf tissues. The genes related to betulinic acid biosynthetic pathway exhibited an expression rate different from other triterpenoid pathway genes, which could be observed in the leaves and roots of control plants and the roots of salt-treated plants. Furthermore, results showed that these two SQS genes have different expression rates due to different plant tissues (roots and leaves) and stress conditions. Importantly, in contrast to previous reports, we detected the glycyrrhizin in leaf tissues. This result may indicate the presence of a different genetic background in native Iranian licorice germplasm. Keywords Gene expression. Glycyrrhiza glabra. Glycyrrhizin. Quantitative real-time PCR. Secondary metabolites Abbreviations bAS β-Amyrin synthase CAS Cycloartenol synthase CYP88D6 β-Amyrin 11-oxidase CYP93E6 β-Amyrin 24-hydroxylase FDP Farnesyl diphosphate GA3 Gibberellic acid HPLC High-performance liquid chromatography LUS Lupeol synthase MeJA Methyl jasmonate OSCs Oxidosqualene cyclases QRT-PCR Quantitative reverse transcription PCR SQS Squalene synthase
Protein domains with similarity to plant strictosidine synthase-like (SSL) sequences have been un... more Protein domains with similarity to plant strictosidine synthase-like (SSL) sequences have been uncovered in the genomes of all multicellular organisms sequenced so far and are known to play a role in animal immune responses. Among several distinct groups of Arabidopsis thaliana SSL sequences, four genes (AtSSL4-AtSSL7) arranged in tandem on chromosome 3 show more similarity to SSL genes from Drosophila melanogaster and Caenorhabditis elegans than to other Arabidopsis SSL genes. To examine whether any of the four AtSSL genes are immune-inducible, we analysed the expression of each of the four AtSSL genes after exposure to microbial pathogens, wounding and plant defence elicitors using real-time quantitative RT-PCR, Northern blot hybridisation and Western blot analysis with antibodies raised against recombinant AtSSL proteins. While the AtSSL4 gene was constitutively expressed and not significantly induced by any treatment, the other three AtSSL genes were induced to various degrees by plant defence signalling compounds, such as salicylic acid, methyl jasmonate and ethylene, as well as by wounding and exposure to the plant pathogens Alternaria brassicicola and cucumber mosaic virus. Our data demonstrate that the four SSL-coding genes are regulated individually, suggesting specific roles in basal (SSL4) and inducible (SSL5-7) plant defence mechanisms.
OBJECTIVE: HDACs are among transcriptional regulatory elements that regulate key features of prol... more OBJECTIVE: HDACs are among transcriptional regulatory elements that regulate key features of proliferation and differentiation in all cell types including cancerous. They may also interfere in such stages of cancer development as migration, invasion, multi-drug resistance and angiogenesis. Proven information about HDAC1 role in development of bladder cancer is limited only to cell lines in vitro. The lack of a comprehensive clinical in vivo study led us to evaluate HDAC1 expression in human clinical specimens. METHODS: We analyzed a large group of bladder cancer patients. The presence of hHDAC1 mRNAs were tracked using specifi c HDAC1 primers in cancer samples and the quantity of HDAC1 transcripts were quantifi ed using real time qPCR method and was compared to those of normal bladder samples from healthy patients. RESULTS: HDAC1 mRNA expression was signifi cantly elevated in Bladder cancer specimens. To our knowledge, this result is the fi rst, showing an elevation in vivo in HDAC1 mRNA levels in clinically cancerous tissue of patients with bladder cancer. CONCLUSIONS: We conclude that hHDAC1 overexpression might be implicated in bladder cancer tumorigenesis and that the over-expressed HDAC1 mRNA might be a potential diagnostic marker and, a target for treatment of bladder cancer using HDACi-drugs in future (Tab. 2, Fig. 2, Ref. 30).
Strictosidine synthase is a key enzyme in the biosynthesis of plant alkaloids. The homologous of ... more Strictosidine synthase is a key enzyme in the biosynthesis of plant alkaloids. The homologous of the strictosidine synthase domain has been found in different organisms such as Arabidopsis thaliana. Bioinformatics and expression studies on one of the strictosidine synthase-like gene (SSL6) in Arabidopsis plants showed that gene responded to the signaling molecules, fungal and viral pathogens. The functional role of SSL6 has been further studied using an Arabidopsis knockout mutant with a T-DNA inserted into exon 1. The plants were inoculated with Alternaria brassicicola, which had incompatible interaction with A. thaliana. The fungal biomass was quantified by quantitative real-time PCR and spore count techniques. The assays indicated that T-DNA mediated ssl6 mutant was more susceptible than Col-0 genotype. The chlorophyll content and the size of necrotic lesions were significantly smaller in the ssl6 mutant compared with the Col-0. This is the first report with reference to the functional analysis of SSL6 suggesting that might be SSL6 plays an important role, among other factors, in plant defense against A. brassicicola.
Genetic Engineering and Biosafety Journal, Oct 10, 2012
enescence is a final developmental stage of leaf, which is very important as genetic and physiolo... more enescence is a final developmental stage of leaf, which is very important as genetic and physiological aspects. Many genes are activated at this stage and most of them show remarkable transcript. The function of senescence is to control regulatory physiological changes. This include cessation of photosynthesis, chloroplast degradation, chlorophyll loss and protein breakdown. Senescence can be initiated by a wide variety of different internal and external factors, as well as being an essential part of plant development; senescence in leaves is also induced prematurely by a number of different environmental stresses. Since plants cannot escape from adverse environmental conditions senescence is one mechanism that plants have evolved to cope with such problems. Interestingly, senescence could be induced in plants even after harvest. This phenomenon is observed in such vegetables like broccoli, lettuce and cabbage. Many different senescence-enhanced genes have been isolated, characterized and cloned. Expression analysis of these genes showed a broad range of expression some time before phenotypic changes to last stage of senescence. So it seems that many signaling pathways should be involved in this process.
Licorice is the roots and stolons of Glycyrrhiza uralensis which have several chemical compounds.... more Licorice is the roots and stolons of Glycyrrhiza uralensis which have several chemical compounds. Triterpene saponins such as glycyrrhizin and glycyrrhetinic acid and flavonoids like liquiritin, isoliquiritigenin and glabron are main compounds detected in liquorice root. The plant's major constituent is a glycyrrhizin. The β-amyrin 11-oxidase catalyzes the sequential two-step oxidation of β-amyrin in C-11 to produce 11-oxo-β-amyrin, a possible biosynthetic intermediate between β-amyrin and glycyrrhizin. In this study, the total RNA was extracted from licorice roots and cDNA synthesis, then PCR products were cloned into pTZ57R/T vector. Sequencing confirmed piece length of 1482 bp that encodes a protein of 493 amino acid residues. The results of alignment showe d 99% similarity to βamyrin sequence of Glycyrrhiza uralensis. Subcellular studies using Softberry and Psort software showed that the activity of this protein is in endoplasmic reticulum. Moreover the protein has a signal peptide and is targeted to the secretory pathway. The results of phylogenetic tree determined most similar amino acid sequence to the CYP88D subfamily of cytochrome P450. These findings can be used for nucleotide or protein manipulation and transformation.
The entomopathogenic fungi-like Beauveria bassiana must penetrate via the integument of an insect... more The entomopathogenic fungi-like Beauveria bassiana must penetrate via the integument of an insect to reach the hemocoel. Since proteins are the molecules responsible for integument strength in insects, the proteins must synthesise the cuticle degrading proteases which will then enable the proteases to penetrate. It is important to determine the biochemical properties of these proteases so that fungal virulence can be better understood. In the current study, a recently collected isolate of B. bassiana, namely AM-118, was inoculated in liquid media containing 0.5% of Andrallus spinidens Fabricus cuticle to obtain specific proteases. The crude samples were purified via a three step process using ammonium sulfate, Sepharyl G-100, and DEAE-Cellulose Fast Flow. The results revealed two proteases known as subtilisin-like (Pr1), and trypsin-like (Pr2), with the molecular weights of 105 and 103 kDa. The optimal pH and temperature values were found to be 8 and 35°C for Pr1 and 8 and 40°C for Pr2, respectively. Inhibitors like AEBSF, EDTA, TPCK, and phenanthroline significantly affected proteolytic activities. Here, we reported two fungal proteases by high molecular weight from an Iranian isolate of B. bassiana. These findings will help us to better understand fungal virulence against insects.
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