Research Journal of Biotechnology

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Research Journal of Biotechnology





Genetic differentiation between Awassi and Merino sheep breeds using Microsatellites

Al-Atiyat Raed Mahmoud

Genetic differentiation of Awassi sheep of Jordan and Merino sheep from Australia was investigated using six microsatellite markers (MS). Three studied populations of each Awassi and Merino revealed high genetic variation. Awassi middle population was most genetically variable which might be a result of gene flow. However, lower genetic variation was observed in Merino populations which might due to breeding practices under which selection took place. Overall results indicated an evolutionary distinction of Awassi sheep from Merino sheep. Furthermore, the individual admixture assigned them to predefined population resulted in successful genetic differentiation of Awassi sheep from Merino sheep. On the other hand, the average admixture proportion of Awassi and Merino individuals was low (20%) indicating non-admixed individuals in general. This result is probably due to common ancestry origin and recent migration. The assignment analysis was grouping individuals in clusters belonging to each separated population rather than being mixed population. Finally, genetic distances and phylogenetic tree evolutionary separated Awassi from Merino flocks. These results are in agreement with known history of the populations in regards to their far away geographical location and their long evolutionary distinct. To sum up, evidences of recent crossbreeding were not observed between Awassi and Merino sheep in Jordan.

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Molecular Characterization and Tissue Expression of the FSP27 Gene in Wujin Pigs

Pan Hongbin, Zhao Sumei, Jiang Jiawei, Huang Ying, Yang Minghua, Sun Yongke, Gao Shizheng and Qin Guixin

Fat-specific protein 27 (FSP27) could be a potential biomarker promoting neutral lipid storage and thereby a candidate gene for the regulation of the intramuscular fat (IMF) deposition in pigs. In this study, the cloning and comparison of the coding domain sequence (CDS) and the deduction of amino acids sequence of FSP27 gene in longissimus dorsi muscle between Wujin and Landrace pigs suggest that the CDS of FSP27 gene is 747 bp encoding for 248 amino acids. One silent polymorphism (C→T) occurred in 549th nucleotide in the CDS of the FSP27 gene in Wujin pigs. Secondary structure analysis of FSP27 deduced amino acids found 67 helixes, 49 strands, 132 coils and 8 transmembrane helices. The FSP27 was localized in the mitochondria. Moreover, the FSP27 contained 6 exons in which the sequence lengths were 33, 78, 154, 159, 188 and 682bp, respectively. The 3'-UTR region of FSP27 gene contained potential regulatory sequences for some miRNA, for example hsa-miR-491-5p and hsa-miR-4510. The promoter region of FSP27 gene exhibited binding sites of AML-1a, NF-kappa-beta and SRY transcription factors. The expression level of the FSP27 mRNA was significantly higher in longissimus dorsi muscle of Wujin pigs than Landrace pigs (P<0.05). The different expression levels of the FSP27 mRNA in longissimus dorsi muscle of pigs may be related to the variation of the IMF deposition.

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A Study of Freezing Behavior for Cryopreservation in Walnut Winter Buds

Choudhary R., Malik S. K. and Chaudhury R.

The freezing behavior of walnut dormant bud was examined using Scanning electron microscopy (SEM) and Light microscopy. All living cells in bud tissues showed distinct shrinkage without intracellular ice formation as a result of slow cooling (5 oC/day) of dormant buds to -30oC through SEM. However, the recrystallization of these slowly cooled tissue cells after LN and then rewarming to -10oC, confirmed that some of the cells in the apical meristem lost freezable water with slow cooling to -30oC indicating adaptation of these cells by deep super cooling. Deep supercooling could not occur in dormant bud primordia if xylem vessels formed a continuous conduit connecting the dormant bud primordia with the remainder of the plant. If xylem continuity was established, ice could propagate via the vascular system and nucleate the water. It is concluded that no extracellular ice crystals accumulated in such tissues containing deep supercooling.

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Screening Selection Identification Production and Optimization of Bacterial Lipase isolated from Industrial Rejection of Gas Station

Larbidaouadi K., Benattouche Z. and Abbouni B.

Production of extracellular lipase in submerged culture of Bacillus licheniformis has been investigated. Bacterial Lipase producers were isolated from industrial rejection of gas station. One of the sixty isolated strain exhibiting a higher lipase activity was selected and identified based on morphological and biochemical characteristics. The effect of incubation time, medium pH, temperature, carbon source and nitrogen source for the lipase production was studied. The lipase production was maximum at pH 8, temperature 40°C and incubation time 48 hours by the lipase producing bacteria Bacillus licheniformis. With a selected carbon source, olive oil and glucose were suitable substrate to maximize lipase production (1.5 U/ml). The optimized concentration of olive oil and glucose was 1%. The effect of nitrogen source on lipase production indicated that the yeast extract was suitable substrate for accelerating lipase production (1.47 U/ml).

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Design and characterization of immobilized urease in alginate bead ‏

Danial Enas N., Hamza Amal H. and Mahmoud Rasha H.

Enzymes have been used in many practical applications, particularly in biomedical and biotechnological fields, through immobilization on a variety of supports. In this study, we evaluate the biological importance of immobilized urease enzyme over the free urease. The alginate was used as a support material for urease immobilization. Generally, the immobilization of urease in alginate gel showed a marked increase in Km and Vmax. However, the thermal stability of the immobilized urease was much higher than that of the free enzyme. The rate of thermal inactivation of the immobilized enzyme decreased due to entrapment in gel matrix. In addition, the activity of the immobilized urease was more stable in retention than that of the free enzyme during the storage in solution. Even though the activity of the immobilized enzyme was lower in comparison with the free enzyme, the enhancement of thermal and storage stability highlights the value of alginate gel as a support for enzyme immobilization. A stable immobilized system and long storage life are convenient for applications that would not be feasible with a soluble enzyme system. These results highlight the technical and biochemical benefits of immobilized urease over the free enzyme.

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Bioprospecting for extracellular hydrolytic enzymes from culturable thermotolerant bacteria isolated from Manikaran thermal springs

Suman Archna, Verma Priyank, Yadav Ajar Nath and Saxena Anil Kumar

Microbial enzymes play a major role in hydrolysis of lignocellulosic compounds to fermentable sugars. In order to isolate thermotolerant bacterial isolates in Manikaran hot springs having potential to produce extracellular hydrolytic enzymes were analyzed using different nutrient combinations. Of 120 isolates, twenty strains showed hydrolytic enzymes production at >70 C. Phylogenetic analysis of positive strains, based on 16S rDNA sequences indicated that isolates were clustered within 48% Proteobacteria, 33% Firmicutes and 19% Actinobacteria. Evaluation of hydrolytic enzymes production under submerged and solid state fermentation was done using paddy straw as sole carbon source. Out of twenty, seven bacterial isolates were found to be novel and efficient for the production of hydrolytic enzymes. A large variation was observed among different isolates for hydrolytic enzymes production. Seven isolates Lysinibacillus sp., Enterobacter cloacae, Rhodococcus qingshengii, Paenibacillus pabuli, Bacillus pumilus, Micrococcus indicus and Pseudomonas fragi based on their maximum production on all different temperature especially 70°C were evaluated in terms of enzyme properties and kinetics. The enzyme of above seven isolates is active over broad range of high temperature. Such thermo stable isolates have potential to be used to develop as consortia for bioconversion of lignocellulosic residue to fermentable sugars preferably at high temperature.

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Evaluation of Forage Production and Tissue Culture Efficiency of Two Teff Grass (Eragrostis Teff) Cultivars

Lee Sang-Hoon, Lee Dong-Gi and Lee Ki-Won

The objective of this study was to evaluate the forage quality and characteristics of two Korean teff grass cultivars, Tiffany and Emerald and to evaluate culture responses. These were determined through a field experiment conducted at the Agronomic Research Area, Grassland and Forages Division, National Institute of Animal Science, Cheonan, Korea, during 2013. The cultivars were arranged in a randomized, complete block design in triplicate. Significant differences were observed among the cultivars with regards to forage yield and quality with the variety Tiffany having significantly higher forage and quality parameters. Experiments were conducted using optimized culture medium to determine optimal callus induction and plant regeneration. The callus induction and plant regeneration of the two cultivars of teff grass differed with higher overall plant regeneration rates observed in the Tiffany cultivar

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Ginsenoside Rg3 sensitizes colon cancer cells to DNA damage reagents by inhibiting WEE1 kinase expression

Chen Wenchao and Zhang Xiefu

Recent research indicates that ginsenoside Rg3 can sensitize colon cancer cells to DNA damage reagents. But the molecular mechanisms of ginsenoside Rg3 function remain elusive. In this study we report the new mechanism by which ginsenoside Rg3 sensitize tumor cells to DNA damage reagents. Cell viability assays indicated that ginsenoside Rg3 sensitized WiDr, SW948, COLO205 colon cancer cell lines to cisplatin and 5-FU in dose-dependent manner. Real-time PCR results showed that ginsenoside Rg3 decreased WEE1 expression in dose- and time-dependent fashion. WEE1 plasmid was also constructed and transfected into tumor cell to overexpress WEE1. The results indicated that overexpression of WEE1 in colon cancer cell lines suppressed the sensitization effect of ginsenoside Rg3 to cisplatin and 5-FU. Our results indicate that ginsenoside Rg3 has the capability of sensitizing colon cancer cells to therapeutic DNA damage reagents by suppressing WEE1 expression and can be a good option of adjuvant chemotherapy for colon cancer patients.

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Molecular characterization of mulberry (Morus spp.) genotypes by Inter-simple Sequence Repeat markers

Wani Shabir A., Bhat M. Ashraf, Malik G. N., Mir M. R. and Iqbal Javid

Mulberry (Morus spp.) is an important tree crop being exploited for feeding the silk producing insect Bombyx mori L. The characterizations of germplasm based on genetic diversity are efficiently done by DNA markers for crop improvement in mulberry. In the present investigation seventeen mulberry genotypes were analyzed for their genetic diversity studies based on morphological and molecular markers and 20 inter-sequence repeat primers were used. Moisture content was recorded highest (73.21 %) in Goshoerami and lowest in Brentul (70.40%) during spring while as in autumn it was recorded (73.13%) in Goshoerami. Moisture retention capacity after 6hrs and 12hrs was found maximum in SKM-33 during both the seasons. A total of 97 alleles were generated across the genotypes. The number of alleles per loci generated varied from 4 (830) to 8 (UBC-820) with an average of value of 4.85. Polymorphism information content values varied from 0.735 (830) to 0.867(UBC-820) with an average of 0.776 per loci. The similarity coefficient ranged from 0.65 to 0.86 with an average value of 0.75. Least similarity coefficient (0.65) was observed between Kokuso-21 and Kanva-2. Clustering analysis grouped the genotypes into seven clusters. Polymorphism information content (PIC) value, which is reflection of allelic diversity and frequency among the genotypes, varied from 0.735 (830) to 0.867(UBC-820) with an average of 0.776 per loci.

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Skin Anti-inflammatory Activity of Nano-encapsulated Aronia melanocarpa Extracts

Kim Nam Young and Lee Hyeon Yong

70% ethanol extract of Aronia melanocarpa was encapsulated with an edible encapsulant, lecithin, because this extract showed better biological activities than those of a conventional hot water extract. The nano-encapsulation efficiency of the ethanol extract was estimated to be 42.6% with an average diameter of 100-200 nm; the efficiency was higher for an aqueous extract of Aronia melanocarpa than for those of other medicinal plants. The nanoparticles showed a high antioxidant activity of 74.29% of α,α-diphenyl-β-picrylhydrazyl (DPPH) free-radical scavenging activity, while the EE was 67.63% after adding 1.0 mg ml-1. The nanoparticles exhibited 35.6% efficacy in inhibiting hyaluronidase enzyme activities, which were higher than that of the 70% ethanol extract. We observed the lowest nitric oxide (NO) production, 8.02 µM, from RAW 264.7 cells after adding 1.0 mg ml-1 of nanoparticles. After adding the equivalent amount of 70% ethanol extracts, 9.26 µM of NO was produced. In addition to the production of NO from RAW 264.7 cells, the secretion of IL-6 and TNF-α from Jurkat T cells was measured as 3.7 pg ml-1 and 1297.1 pg ml-1, respectively after adding 1.0 mg ml-1 of the nanoparticles. In general, the nanoparticles proved to have better skin anti-inflammation activities than that of the 70% ethanol extract because of their association with high antioxidant activity; this was despite the actual concentrations of the extract in the nanoparticles being only 43% of the crude ethanol extract concentration as a result of the encapsulation efficiency. Real-time confocal microscopy and fluorescence activated cell sorter (FACS) analyses clearly proved that the nanoparticles with lower amounts of the bioactive components can have higher skin anti-inflammatory activity than that of the ethanol extract due to their fast penetration through the cell membrane within 40 min after the inoculation of the cells.

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Biodegradation of petroleum oil by mangrove fungi from Saudi Red Sea Coast

Ameen Fuad, Moslem Mohamed and Hadi Sarfaraz

Mangrove sediments were collected from different localities on the south Red Sea Coast of Saudi Arabia. Forty five isolates belonging to 13 genera were recovered and three of these isolates were found to be able to grow in association with petroleum oil as sole carbon source under in vitro conditions. These isolates were identified as Cladosporium sphaerospermum, Eupenicillium hirayamae and Paecilomyces variotii using 18S rDNA gene analysis. The three isolates have high potential to degrade petroleum hydrocarbons by co-cultivation under aeration on a rotary shaker. As compared to the controls, these fungi accumulated significantly higher biomass, produced extracellular enzymes and liberated larger volumes of CO2. These observations with Gas chromatography data confirm that these strains could be interesting to use in future field tests.

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Molecular Characterization of Selected Egyptian Wheat Genotypes by RAPD and ISSR

Mahmoud Mohamed A., Abd-El-Aziz Abeer R. M., Al-Othman Monira R. and Imbaby Ibrahim A.

Random Amplified Polymorphic DNA (RAPD) markers and Inter Simple Sequence Repeat (ISSR) markers were used for genetically characterizing of 11 Egyptian wheat genotypes. 8 RAPD and 8 ISSR markers were used to assessment the genetic diversity. RAPD analysis revealed that 60 out of 107 bands (49.45%) were polymorphic while ISSR analysis revealed that 75 out of 96 bands (60.38%) were polymorphic. The UPGMA dendrogram generated using the similarity matrix produced from the banding patterns obtained with RAPD and ISSR markers exhibited different patterns of cluster analysis. In RAPD analysis, the average genetic similarity generated based on RAPD profiling showed 60.4-92.7 % GS between the tested wheat genotypes while in ISSR analysis, showed 66.5-91.1 % GS. Genetic similarity and dissimilarity among genotypes will be useful for genetic variations and can be used to assist efficient selection in wheat breeding programs aiming at achieving sustainability in wheat production in Egypt.

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Optimization of cold–active lipase production by CALIP1 from deep–sea sediment–derived metagenomic library

Yi Zhiwei, Chan Zhuhua, Yang Fan, Liu Yang and Zeng Runying

A clone, CALIP1, exhibiting lipase activity was selected from the metagenomic library derived from deep–sea sediment. The purified lipase displayed optimal activity at pH 8.0 and 25 °C and maximal stability at 30 °C, indicating that the lipase from CALIP1 is a cold–active enzyme. To enhance the lipase production, the Plackett–Burman design was selected to screen the medium components that significantly influence the production. Three components that influenced the production include olive oil, yeast extract powder and (NH4)2SO4. The optimal concentration of these components was determined by response surface methodology which is based on the central composite design. Under the proposed optimized conditions, the lipase experimental yield (2760 Uml-1) closely matched the predicted yield by the statistical model (2811.84 Uml-1) with R2=0.987. An overall 86.5% increase in lipase yield compared with the original culture medium.

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