جستجوی مقالات مرتبط با کلیدواژه "سلولاز" در نشریات گروه "بیوتکنولوژی و ژنتیک گیاهی"

 Licorice (Glycyrrhiza glabra) is a perennial plant from the Fabaceae family rich in bioactive secondary metabolites used to treat many human diseases. Recent advances in plant tissue culture techniques have shown promising results in improving productivity and allowing the gradual replacement of whole plant culture as a source of valuable secondary metabolites. Today, various tissue culture methods are used to increase the performance of secondary metabolites by strengthening plant defense and stimulating stress response in plant cells with the help of elicitors. Plant pathogenic fungi produce cellulase, which is used as an elicitor to stimulate the production of secondary metabolites in plant cell suspension culture conditions. This research aimed to study the effect of cellulase enzyme obtained from Aspergillus nigar fungus on the amount of antioxidant activity (DPPH method) and antioxidant enzyme activity in licorice cell suspension culture conditions. 

 Licorice seeds were collected from the Semirom region, Isfahan province, Iran. Seeds were cultured in the ½ MS medium after disinfection. In order to induce callus, the hypocotyl and cotyledon were cultured in MMS medium containing growth regulators (2 mg/L BA and 0.5 mg/L NAA) with 3% sucrose and 0.7% agar. Then 0.5 g of fibrous callus produced under a sterile hood was transferred to Erlenmeyer flasks containing 50 cc of MMS liquid culture medium containing 2 mg/liter of BA hormone and 0.5 mg/liter of NAA hormone and was placed in a shaker incubator with a speed of 150 rpm, a temperature of 25oC and darkness. On the 19th day after cultivation, cellulase enzyme with a 200 μg/ml concentration was added to each Erlens under a sterile hood. Then, harvesting was done at zero-time intervals (control) 24, 48 and 72 hours after adding the elicitor. Late cultured calli were also considered as a treatment. Statistical analysis was carried out using a completely randomized design with three replications. Data were collected for DPPH and all antioxidant enzyme activities such as catalase, superoxide dismutase, polyphenol oxidase, ascorbate peroxidase and guaiacol peroxidase. A regression analysis was used to determine the relationship between DPPH and antioxidant enzyme activities at the time interval after adding the elicitor. 

 The effect of cellulase enzyme on all traits except superoxide dismutase was significant (P<0.01). The activity of antioxidant enzymes and antioxidant activity (DPPH method) within 72 hours of applying treatment showed the most significant increase compared to the control. Correlation between traits showed that catalase enzyme had a positive and significant correlation with polyphenol oxidase and guaiacol peroxidase enzymes. Similarly, the guaiacol peroxidase enzyme positively correlated with the polyphenol oxidase enzyme (P<0.01). Antioxidant activity (DPPH method) also showed a positive and significant correlation with catalase, polyphenol oxidase and catalase enzymes. The result of simple regression analysis using DPPH and all antioxidant enzyme activities as dependent variables and the time interval after adding the elicitor as independent variables showed that all DPPH and all enzyme activities (except superoxide dismutase and ascorbate peroxidase) had increased by 72 hours after adding the elicitor.

 The use of cellulase enzyme derived from Aspergillus nigar fungus in the conditions of cell suspension culture increases the antioxidant activity (DPPH method) and the activity of oxidant enzymes. Therefore, it was concluded that the cellulase enzyme caused cell stress in the conditions of cell suspension, and this stress caused the stimulation of the plant's defense mechanisms, such as antioxidant enzymes. A positive and significant correlation was observed between the antioxidant activity and some antioxidant enzymes, such as polyphenol oxidase and catalase, which indicates the positive effect of the fungal elicitor in stimulating the production of secondary metabolites and increasing the potential of inhibiting free radicals in licorice plant cell suspension culture conditions. The DPPH and all enzyme activities (except superoxide dismutase and Ascorbate Peroxidase) were increased 72 hours after adding the elicitor.

In order to identify the endophytic fungi of Azarshahr red onion variety, sampling was conducted from the main cultivation areas of this plant in East Azarbaijan province. A total of 360 fungal isolates were obtained from different parts of onion (root, base, scale, flowering stem and leaf). The isolates were grouped based on their phenotypic characteristics on culture media and comparing the DNA fingerprinting patterns using the ISSR molecular markers. In order to accurate identification of the endophytic fungi, the morphological characteristics were combined with the phylogeny inferred from analysis of ITS-rDNA genomic region sequence. Examined endophyte isolates were belonged into eight genera and 12 species namely Alternaria atrum, A. tenuissima, Aspergillus niger, A. kevei, Fusarium equiseti, F. oxysporum, F. proliferatum, F. solani, Mortierella alpina, Setophoma terrestris, Stachybotrys chartarum and Trichoderma longibrachiatum. The results of the inhibitory effect experiments of the selected endophyte Trichoderma longibrachiatum strain ASh268 on the growth of several pathogenic fungal isolates including Rhizoctonia solani, Fusarium oxysporum, Fusarium solani and Setophoma terrestris showed that this strain has a good ability to produce cellulase enzyme in carboxymethyl cellulose medium and significantly reduces the growth of all the above pathogens on the culture media. To our knowledge, this is the first report of Aspergillus kevei for the funga of Iran. Also, this research is the first study on the isolation and identification of endophytic fungi of red onion verity in its major cultivation areas in Iran.

Small and powerful promoters play a key role in recombinant protein expression in different hosts including Pichia pastoris. In the present study, expression of cellulase beta endoglucanase CMC3 gene was studied under the control of the alcohol oxidase 1 (AOX1) and methanol oxidase (MOX) promoters, with the methanol feeding, in the yeast Pichia pastoris. To avoid the gene dosage effect, colonies harbored single copy of target constructs were screened by Real-Time PCR method. The results of enzymatic assay, zymogaphy and SDS-PAGE for the culture of single copy harboring transformants showed powerful, comparable expression of CMC3 under the control of both MOX and AOX1 promoters. High-level expression of recombinant CMC3 under control of the MOX promoter indicated that this small, new powerful promoter could be considered as a promising tool for recombinant protein production in the yeast Pichia pastoris.