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کاربرد نوع شرط:
- جایگاه : پژوهشی
- مجله: Progress in Biological Sciences
- نوع مقاله: Journal Article
- کلمات کلیدی: Recombinant protein,Pichia pastoris,Hansenula polymorpha,endoglucanase
- چکیده:
- چکیده انگلیسی: Bioconversion of cellulosic material into bioethanol needs cellulase complex enzymesthat contain endoglucanase, exoglucanase and beta glucosidase. One of the most important organisms that produce cellulases is the filamentous fungi, Trichoderma reesei which able to secrete large amounts of different cellulases. These enzymes are probably the most widely used cellulases industrially, however, the cellulases excreted from fungi are not stable at high pH or high temperatures. In this study methylotrophic yeasts, Pichia pastoris and Hansenula polymorpha were used for the comparative heterologous production of endoglucanase II. Two synthetic egII genes with P. pastoris and H. polymorpha codon preferences were transferred into the yeasts. In addition, both expression vectors contained the pre-pro-sequence of Saccharomyces cerevisiae mating factor alpha to allow secretion of protein. Enzymes characterization demonstrated increasing thermal stability in both recombinants EGII compare with native enzyme from T. reesei and the Hansenula enzyme was more stable than Pichia in higher temperature. Biochemical properties determination on different substrates showed higher binding site affinity in Pichia than Hansenula and native one. We can conclude that P. pastoris and H. polymorpha are appropriate hosts for expression and production of endoglucanase with improved thermal stability.
- انتشار مقاله: 04-12-1391
- نویسندگان: Ali Akbarzadeh,Seyed Omid Ranaei Siadat,Mohammad Reza Zamani,Mostafa Motallebi,Mohammad Barshan Tashnizi
- مشاهده
- جایگاه : پژوهشی
- مجله: Progress in Biological Sciences
- نوع مقاله: Journal Article
- کلمات کلیدی: Recombinant protein,Pichia pastoris,Hansenula polymorpha,endoglucanase
- چکیده:
- چکیده انگلیسی: Bioconversion of cellulosic material into bioethanol needs cellulase complex enzymesthat contain endoglucanase, exoglucanase and beta glucosidase. One of the most important organisms that produce cellulases is the filamentous fungi, Trichoderma reesei which able to secrete large amounts of different cellulases. These enzymes are probably the most widely used cellulases industrially, however, the cellulases excreted from fungi are not stable at high pH or high temperatures. In this study methylotrophic yeasts, Pichia pastoris and Hansenula polymorpha were used for the comparative heterologous production of endoglucanase II. Two synthetic egII genes with P. pastoris and H. polymorpha codon preferences were transferred into the yeasts. In addition, both expression vectors contained the pre-pro-sequence of Saccharomyces cerevisiae mating factor alpha to allow secretion of protein. Enzymes characterization demonstrated increasing thermal stability in both recombinants EGII compare with native enzyme from T. reesei and the Hansenula enzyme was more stable than Pichia in higher temperature. Biochemical properties determination on different substrates showed higher binding site affinity in Pichia than Hansenula and native one. We can conclude that P. pastoris and H. polymorpha are appropriate hosts for expression and production of endoglucanase with improved thermal stability.
- انتشار مقاله: 04-12-1391
- نویسندگان: Ali Akbarzadeh,Seyed Omid Ranaei Siadat,Mohammad Reza Zamani,Mostafa Motallebi,Mohammad Barshan Tashnizi
- مشاهده
- جایگاه : پژوهشی
- مجله: Mechanics of Advanced Composite Structures
- نوع مقاله: Journal Article
- کلمات کلیدی: Mathematical Modeling,Free vibration,Thermal buckling,Perforated plate,Heaviside function
- چکیده:
- چکیده انگلیسی: This artcile is concerned with thermal buckling and thermal induced free vibration analyses of PCPs (perforated composite plates) with simply supported edges applying a mathematical model. The stiffness and density of PCP are defined locally using Heaviside distribution functions. The governing equations are derived based on CLPT. The present solution gives reasonable results in comparison with the few literatures. In order to inspect the structural behaviour of PCPs subjected to initial thermal loads, many parametric studies have been carried out. Results indicated that the presence of perforations has a significant effect on thermal buckling and thermal induced fundamental frequency.
- انتشار مقاله: 01-09-1397
- نویسندگان: Sina Soleimanian,Ali Davar,Jafar Eskandari Jam,Mohammad Reza Zamani,Mohsen Heydari Beni
- مشاهده
- جایگاه : پژوهشی
- مجله: Mechanics of Advanced Composite Structures
- نوع مقاله: Journal Article
- کلمات کلیدی: Free vibration,cutout,GFRP Plates,Free Boundaries
- چکیده:
- چکیده انگلیسی: This study explored the free vibration problem in relation to glass fiber reinforced polymer (GFRP) plates with central cutouts and free boundaries using theoretical, experimental, and numerical methods. The theoretical formulations were derived from the classical lamination plate theory. The rectangular cutout was mathematically modeled into the stiffness matrix of the plate by multiplying Heaviside distribution functions. The theoretical values for the fundamental frequency were obtained by solving the standard eigenvalue problem, and the theoretical solution was validated by comparison to the literature. Modal testing was performed in the laboratory. For additional validation, the accuracy of theoretical and experimental results was checked using the finite element method and ABAQUS. The results of all three methods agreed; thus, the applicability of the Heaviside functions to stiffness modeling of structures with cutouts was proven. It was also observed that the fundamental frequency decreased when cutout size increased.
- انتشار مقاله: 15-05-1396
- نویسندگان: Sina Soleimanian,Ali Davar,Reza Azarafza,Jafar Eskandari Jam,Mohammad Reza Zamani
- مشاهده
- جایگاه : پژوهشی
- مجله: Iranian Journal of Biotechnology
- نوع مقاله: Journal Article
- کلمات کلیدی: Cis-acting elements,Inducible promoter,Selectable marker gene,Self-excision system
- چکیده:
- چکیده انگلیسی: Abstract Background: Selectable marker gene (SMG) systems are critical for generation of transgenic crops. Transgenic crop production Background: Selectable marker gene (SMG) systems are critical for generation of transgenic crops. Transgenic crop production without using SMG is not economically feasible. However, SMGs are non-essential once an intact transgenic plant has been established. Elimination of SMGs from transgenic crops both increases public acceptance of GM crops and prepares gene stacking possibility for improvement of complex traits. Synthetic inducible promoters provide an efficient and flexible strategy to regulate transgene expression. Objectives: This study aimed to construct a transformation vector based on Cre/loxP recombination system to enhance efficiency of SMG-free transgenic plant production followed by post-excision expression of gene of interest in transgenic plants by a pathogen inducible promoter. Materials and Methods: In pG-IPFFDD-creint-gusint construct, cre recombinase and selectable marker gene (nptII) cassettes were placed between the two loxP recognition sites in direct orientation. Seed-specific Napin promoter was used for regulation of Cre expression in transgenic seeds. In the construct, loxP flanked sequence containing nptII and recombinase cassettes, located between a pathogen inducible promoter containing FFDD cis-acting elements and b-glucuronidase coding region. The cunstuct was transformed into Nicotiana tabaccum via Agrobacterium-mediated transformation. Results: The results showed that both cre and nptII excision occurs in T1 progeny tobacco plants through seed-specific cre expression. The excisions were confirmed by methods activation of the gus gene, germination test on kanamycin-containing medium and molecular analysis. Inducibility of gus expression by FFDD-containing promoter in T1 leaf tissues was confirmed by histochemical Gus staining assay. Conclusions: The established system is not only an efficient tool for marker gene elimination but also provides possibility for inducible expression of the transgene.without using SMG is not economically feasible. However, SMGs are non-essential once an intact transgenic plant has been established. Elimination of SMGs from transgenic crops both increases public acceptance of GM crops and prepares gene stacking possibility for improvement of complex traits. Synthetic inducible promoters provide an efficient and flexible strategy to regulate transgene expression. Objectives: This study aimed to construct a transformation vector based on Cre/loxP recombination system to enhance efficiency of SMG-free transgenic plant production followed by post-excision expression of gene of interest in transgenic plants by a pathogen inducible promoter. Materials and Methods. In pG-IPFFDD-creint-gusint construct, cre recombinase and selectable marker gene (nptII) cassettes were placed between the two loxP recognition sites in direct orientation. Seed-specific Napin promoter was used for regulation of Cre expression in transgenic seeds. In the construct, loxP flanked sequence containing nptII and recombinase cassettes, located between a pathogen inducible promoter containing FFDD cis-acting elements and β-glucuronidase coding region. The cunstuct was transformed into Nicotiana tabaccum via Agrobacterium-mediated transformation. Results: The results showed that both cre and nptII excision occurs in T1 progeny tobacco plants through seed-specific cre expression. The excisions were confirmed by methods activation of the gus gene, germination test on kanamycin-containing medium and molecular analysis. Inducibility of gus expression by FFDD-containing promoter in T1 leaf tissues was confirmed by histochemical Gus staining assay. Conclusion: The established system is not only an efficient tool for marker gene elimination but also provides possibility for inducible expression of the transgene.
- انتشار مقاله: 17-09-1393
- نویسندگان: Shiva Hamzeh,Mostafa Motallebi,Mohammad Reza Zamani,Zahra Moghaddassi Jahromi
- مشاهده
- جایگاه : پژوهشی
- مجله: Iranian Journal of Biotechnology
- نوع مقاله: Journal Article
- کلمات کلیدی: Trichoderma,Real-Time Polymerase Chain Reaction,Chitinase,Sclerotinia sclerotiorum
- چکیده:
- چکیده انگلیسی: Backgoround: Plant diseases, caused by a wide range of phytopathogenic fungi, could be managed using of Trichoderma sp, as a biocontrol agent. Cell wall degrading enzymes like chitinase from T. harzianum are important means for fungal pathogen inhibition. Overexpression of these chitinase enzymes can improve the antagonistic potential of Trichoderma sp. strains. Objectives: This study aimed to produce a new enhanced biocontrol system of Trichoderma harzianum, overexpressing chit42 gene. The improved T. harzianum could be an appropriate biocontrol agent for controlling the stem rot disease of canola caused by Sclerotinia sclerotiorum. Materials and Methods: T. harzianum protoplast cotransformation was carried out by pLMRS3-Chit42 and p3SR2 plasmids. The transformants were selected based on their growth on colloidal chitin containing medium. The improvement of transformants was investigated by quantification of mRNA using real-time quantitative polymerase chain reaction (RT-PCR) and measurement of chitinase activity in the medium containing colloidal chitin as the carbon source. Furthermore, the antagonistic activity of transformants against S. sclerotiorum was assessed by dual culture experiments. Results: The overexpressing transformants of Chit42 displayed higher levels of chitinase activity to inhibit S. sclerotiorum growth compared with the wild type. The results indicated that the value of the chitinase activity (126.42 + 0.07 U/mL) of Chit42-9 increased 64.17 fold. Transcriptomic analysis demonstrated that Chit42-9 transformant expressed 5.2 fold of Chit42 transcript as compared with the parent strain. Biocontrol inhibition of this transformant was 4.98-fold more compared with the non-transformant type. Conclusion: The improved Chit42-9 transformant can be used for biocontrolling S. sclerotiorum, cause of stem rot disease in canola.
- انتشار مقاله: 05-05-1392
- نویسندگان: Mojegan Kowsari,Mohammad Reza Zamani,Mostafa Motallebi
- مشاهده
- جایگاه : پژوهشی
- مجله: Iranian Journal of Biotechnology
- نوع مقاله: Journal Article
- کلمات کلیدی: Canola,Sclerotinia sclerotiorum,Secale cereal,Thaumatin-like proteins,Transgenic plant
- چکیده:
- چکیده انگلیسی: Canola (Brassica napus L.) is an important oilseed crop. A serious problem in cultivation of this crop and
yield loss, are due to fungal disease stem rot caused by Sclerotinia sclerotiorum. The pathogenesis-related
(PR) proteins have the potential for enhancing resistance against fungal pathogen. Thaumatin-like proteins
(TLPs) have been shown to have antifungal activity on various fungal pathogens. In this study, the tlp gene
isolated from cereal rye (Secale cereal L.) was introduced into canola plants. The amplified DNA fragment
(about 500 bp) was analyzed and confirmed by restriction pattern and cloned into pUC19 and designated as
pUCNG1. Comparison of the cloned fragment with the DNA sequence indicated that this gene contains no
intron. The tlp gene was predicted to encode a protein of 173 amino acids with an estimated molecular mass
of 17.7 kDa. The deduced amino acid sequence of TLP showed a significant sequence identity with TLP
from S.cereal and other plants. We used a transgenic over-expression approach in order to investigate antifungal activity of expressed TLP on Sclerotinia sclerotiorum. TLP was overexpressed under the CaMV35S
constitutive promoter in (Brassica napus, R line Hyola 308). Transformation of cotyledonary petioles was
achieved via Agrobacterium tumefaciens LBA4404. The insertion of transgene was verified by the polymerase
chain reaction (PCR) and genomic DNA dot blotting. Antifungal activity was detected in transgenic
canola lines using detached leaf assay. The size of lesions induced by S. sclerotiorum in the leaves of
transgenic canola was significantly retarded when compared to that detected in non-transgenic plants.
- انتشار مقاله: 13-01-1391
- نویسندگان: Akram Zamani,Mostafa Motallebi,Parisa Jonoubi,Nayere Sadat Ghafarian-Nia,Mohammad Reza Zamani
- مشاهده
- جایگاه : پژوهشی
- مجله: Iranian Journal of Biotechnology
- نوع مقاله: Journal Article
- کلمات کلیدی: Brassica napus,cis-acting element,Synthetic promoter,Pathogen-inducible,Fungal elicitor,Reporter gene,Sclerotinia sclerotiorum
- چکیده:
- چکیده انگلیسی: Sclerotinia sclerotiorum is a phytopathogenic fungus which causes serious yield losses in canola. A pathogen inducible-promoter can facilitate the production of Sclerotinia-resistant transgenic canola plants. In
this study, the “gain of function approach” was adopted for the construction of a pathogen-inducible promoter.
The synthetic promoter technique was used, which involved the insertion of the dimerized form of the cisacting
element (F) upstream of the minimal CaMV 35S promoter, which drives the expression of the β-glucronidase
(GUS) gene. The pGFF construct containing this synthetic promoter (SynP-FF) was used for stable transformation of the canola plant. Fluorometric GUS expression analysis indicated that the SynP-FF promoter
is responsive to methyl jasmonate and S. sclerotiorum treatments. The results of histochemical GUS
expression patterns showed strong reporter expression in leaf, flower and stem tissues of canola. Hence,
the SynP-FF synthetic promoter, carrying fungal pathogen-inducible features, could be considered as a
valuable tool for controlling the expression of transgenes to improve resistance against the same lifestyle
pathogens.
- انتشار مقاله: 11-10-1389
- نویسندگان: Farhad Shokouhifar,Mohammad Reza Zamani,Mostafa Motallebi
- مشاهده
- جایگاه : پژوهشی
- مجله: Iranian Journal of Biotechnology
- نوع مقاله: Journal Article
- کلمات کلیدی: Trichoderma,Rhizoctonia solani,Chitinase,Potato,b-1,3-glucanase,Fungal disease
- چکیده:
- چکیده انگلیسی: Potato (Solanum tuberosum L.) an agro-economically important food crop in the world, is sensitive to many fungal pathogens including Rhizoctonia solani (AG-3), the causal agent of stem and root rot diseases. Chitinase and glucanase are cell wall degrading enzymes which have been shown to have high antifungal activity against a wide range of phytopathogenic fungi. In the present study, plasmid pBIKE3 harboring a double-gene cassette containing the chitinase (chit42) and b-1,3-glucanase (bgn13.1) genes was constructed. In this construct, the chit42 gene is located between the CaMV 35S promoter and nos terminator derived from pBI121, while the bgn13.1 gene is downstream of a modified CaMV 35S promoter, followed by the nos terminator both of which were derived from the pRTL plasmid. Micro-tubers of potato plants (the Savalan cultivar) were transformed with the pBIKE3 construct via the Agrobacterium delivery system. Integration of these two genes into the potato genome and their expression at the transcriptional level was confirmed by polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR). The radial diffusion assay showed that the heterologous expressed chitinase and glucanase enzymes demonstrated antifungal activity on R. solani (AG-3).
- انتشار مقاله: 31-06-1388
- نویسندگان: Kasra Esfahani,Mostafa Motallebi,Mohammad Reza Zamani,Haleh Hashemi Sohi,Esmat Jourabchi
- مشاهده
- جایگاه : پژوهشی
- مجله: Iranian Journal of Biotechnology
- نوع مقاله: Journal Article
- کلمات کلیدی: Antifungal,activity,Trichoderma atroviride,Chitinase 42,Heterologous expression
- چکیده:
- چکیده انگلیسی: The cDNA from the mycoparasitic fungus Trichoderma atroviride PTCC5220 encoding a 42 kDa chitinase (Chit42) was isolated. The nucleotide sequence of the cDNA fragment as having a 1263 bp open reading frame that encodes a 421 amino acid polypeptide, and a high homology was found with
other reported Chit42 belonging to the Trichoderma sp. The 22 amino acid N-terminal sequence is a putative signal peptide for the possible secretion of the protein. The protein has been expressed and secreted as a mature form in Escherichia coli BL21(DE3) using the pelB leader sequence.
The E. coli strain expressed Chit42 in an active form and secreted the protein into the medium. This recombinant chitinase has been shown to have inhibitory activity on mycelial growth and also, lytic activity on the cell wall of Rhizoctonia solani (AG2-2), causal agent of root rot in sugar beet in vitro.
Expressed chitinase was optimally active at pH 5 and at 40°C. It is thermally stable at 60°C for more than 120 min at pH 5.
- انتشار مقاله: 12-01-1385
- نویسندگان: Mohammad Javad Harighi,Mostafa Motallebi,Mohammad Reza Zamani
- مشاهده