Research Journal of Biotechnology

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Silver Nitrate (AgNO3) induced Micropropagation Protocol for Medicinal Shrub Nyctanthesarbor-tristis L. and assessment of Genetic Integrity through RAPD Markers

Rohilla Ashok K., Sindhu A., Kajla Subhash and Poonia Anil K.

A reproducible protocol for mass multiplication of Nyctanthesarbor-tristis L. was developed by using a two stage culture procedure from nodal and cotyledonary node explants. Amongst the array of media used, MS basal medium supplemented with the combination BAP (0.5 mgl-1) and NAA (0.3 mgl-1) was found most effective for multiple shoot induction with 86.67% explants response and 10.63±0.64 shoots on 42nd day of inoculation. Inclusion of AgNO3 as regeneration enhancer had a promoting effect on multiple shoot induction and amongst various concentrations(1.0-7.0 mgl-1) used, the addition of 3.0 mgl-1 AgNO3 to the optimal concentration of PGRs (MS basal +BAP 0.5 mgl-1 and NAA 0.3 mgl-1) resulted in maximum shoot induction response (96.66%) and maximum number of shoots i.e. 18.67 ±0.13 and 17.90 ±0.09 shoots with 6.28 ±0.09 cm and 5.82 ±0.07 cm mean length obtained from nodal and cotyledonary node explants respectively after 42 days of culture. A six weeks period was found sufficient for in vitro shoots multiplication. Modified MS medium augmented with IBA (2.0 mgl-1)showed highest root induction with a mean of 4.1±0.22 roots per shoot within 21days of culture while the maximum length (4.2 ±0.33 cm) of root was recorded with 3 mgl-1 of IAA. After a period of four weeks, the regenerated plantlets were transferred to pots containing a mixture of sand: soil: vermicompost (1:1:1) and hardened for six weeks before their field transfer with 80±4% survival rate. RAPD analysis of micro- propagated plantlets along with donor plant confirmed the genetic integrity of micro-propagated plants.

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Synthesis and optical characterization of Mn doped CdS and Mn doped CdS/ZnS nanoparticles

Ali Khalilzadegan and Sadjadi Mirabdullah Seyed

The aim of this work was to find an easy and reproducible method to produce CdS, CdS/ZnS core-shell and their Mn doped components. The samples were prepared by co-precipitation method using L-cysteine as a capping agent and characterized by X-ray diffraction (XRD), UV-VIS and photoluminescence (PL) spectroscopy. Calculation of band gap energy using UV-VIS absorption spectra revealed increase of energy band gap in comparison with the bulk CdS (2.4 eV) due to the quantum confinement. The XRD analysis shows that all the samples prepared are in hexagonal structure. The broad peaks of XRD patterns indicate nanocrystalline behavior of the samples. The average nanocrystallite sizes calculated from Scherrer formula were found to be 3-5 nm. The green emission peaks observed for CdS and CdS/ZnS at 532 and 525 nm respectively, were related to the transition from sulphur vacancy donors (VS) to the Valance Band (VB) and the emission band observed at ~590 nm (orange emission) in CdS:Mn and CdS:Mn/ZnS nanoparticles concerned to the emission due to 4T1-6A1 Mn d-d transition. Enhancement of photoluminescence (PL) intensity observed in CdS/ZnS and CdS:Mn/ZnS can be due to the surface defects reducing effect after growth of ZnS shell.

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Development of Real time PCR for Diagnosis of Capripoxviruses

Batra Kanisht, Kumar Aman, Maan S. Narender, Ghosh Arnab, Sunayna, Kumar Naveen and Maan Sushila

Capripoxviruses cause economically important poxviral diseases in cattle, sheep and goats which are reportable to the World Organization for Animal Health. A sensitive, quick and specific confirmatory diagnosis is the foremost requirement for control and eradication of these diseases. In this study, TaqMan and SYBR green based quantitative polymerase chain reaction (qPCR) and conventional PCR assays targeting the P32 gene were developed for the detection of Capripoxvirus DNA in clinical specimens. Both conventional and real time assays used different primer sets. The conventional PCR assay used primer pairs which yielded amplicon of expected size (581 bp). The specificity of amplified P32 gene product was confirmed by its correct size and further by sequence analysis. Real time assay for Capripoxvirus has been developed and validated using SYBR green and TaqMan formats, which yielded a 72 bp product. Although both the conventional and real time assays were highly sensitive and specific for capripoxviruses however, the qPCR in both formats was 1000 times more sensitive than conventional PCR and was able to detect as low as 70 fg of viral DNA. This assay provides a rapid and robust method to detect capripoxviruses following suspicion of disease in endemic or disease-free countries.

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Characterization of multiple extracellular proteases isolated from Bacillus thuringiensis strain Y25

Kati Hatice, Asar Reyhan and Sen Kaya Sultan

Alkaline proteases produced by Bacillus species have applications in several industries such as detergent industries, bioremediation, food industries, leather processing and food products. In this study, Bacillus thuringiensis strain (Y25), isolated from leaf samples, was detected as producing protease in the medium supplemented with casein at 30°C for 64 h. The strain Y25 showed activity over 90% at a wide range of temperature (10-40°C) and at pH’s ranging from 7-9. The optimum pH and temperature for the crude enzyme activity were 8.0 and 30°C respectively. Also, its activity is increased after 4 h of incubation at 30 °C and at pH 8. While in the presence of Mn+2 metal ions the enzyme activity is increased; in the presence of other metal ions (Zn+2, Cu+2) the enzyme activity is decreased. In addition, the enzyme was found to be very stable in hexane solvent. Strain Y25 was shown to produce at least five proteases having molecular weights of about 91, 85, 74, 59 and 44 kDa sizes by using zymogram technique. The purified protease from Bacillus thuringiensis Y25 strain could be very useful to develop industrial processes.

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Isolation, Identification and Molecular Characterization of Cellulolytic Bacteria from Rumen Samples collected from Erzurum Slaughter House, Turkey

Baltaci Mustafa Ozkan and Adiguzel Ahmet

In this study, the isolation, identification and molecular characterization of 36 bacteria samples of Achromobacter, Bacillus, Brevibacillus, Citrobacter, Clostridium, Enterococcus, Escherichia, Exiguobacterium, Lactobacillus, Lactococcus, Macrococcus, Neisseria, Ochrobactrum, Paenibacillus, Shigella,, Staphylococcus, Streptococcus and Streptomyces genera, which show cellulolytic properties, were performed from the rumen samples taken from the slaughterhouses in Erzurum. For this reason, the salt, pH and temperature demands of these isolates, which were considered to be different from each other and which showed cellulolytic properties were determined. Upon the analyses it was determined that the salt ratio in which the test strains could develop best was 2-4% and the pH and temperature range was between 5-9 and 15-37°C respectively. It was also observed that only the strain with the code OZK24 from the cellulolytic bacteria could develop at 13% salt and pH 11 values. In addition, the 16S rRNA sequenceanalyses of the test bacteria were performed and it was observed that the OZK21 strain showed similarity to Macrococus caseolyticus at a rate of 98% and the OZK30 strain showed 97% similarity to Clostridium magnum. According to these data at hand, it was concluded that the potential of the two isolates being new species was high.

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Molecular Characterization of Ghrelin gene in sheep of Saudi Arabia

Mahmoud A.H., Farah M.A., Abou-tarboush F.M., Rady A.M., Alanazi. K M. and Mohammed O.B.

The main role of Ghrelin hormone is the regulation of feed intake, body weight and gastrointestinal motility. It stimulates appetite by acting on the hypothalamic arcuate nucleus. Ghrelin is orexigenic that transmits a hunger signal from the periphery to the central nervous system and is involved in mealtime hunger and meal initiation. The aim of the present study was to characterize the GHRL gene in two Saudi sheep breeds locally named as Najdi and Naeimi. A fragment of 112 bpcomprisingparts of exon 1 and intron 1 of the Ghrelin gene was amplified and sequenced. Our mutations were detected at positions (74,92, 99 and 108) of the intron1. In addition, the four mutations were used to construct different haplotypes, three haplotypes were recovered: H1 (TAAA), H2 (CGGG), and H3 (TGAA). The H1 (TAAA) was the most common haplotype found in Najdi and Naeimi individuals with frequency of 0.73 for both sheep breeds. The H2 haplotype (CGGG) was unique for Najdi sheep with frequency of 0.21. H3 haplotype (TGAA) was represented in Najdi and Naeimi sheep with frequencies of 0.06 and 0.27 respectively. The present study provides basic information to understand the genetic Characterization of local sheep breeds in Saudi Arabia.

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Production Level, Solubility, Proper Folding and Stability Evaluation of Organophosphorus Hydrolase (OPH) enzyme by Expression at Two Systems

Farnoosh Gholamreza, Latifi Ali Mohammad, Khajeh Khosro and Aghamollaei Hossein

The use of the organophosphorus hydrolase (OPH) enzyme to degrade Organophosphate compounds is one of the most frequently used decontamination methods. OPH is an ~36-kDa homodimericmetalloprotein that is found in the membrane of Flavobacterium sp. strain ATCC 27551 and Brevundimonasdiminuta MG and is capable of hydrolyzing a wide range of oxon and thion such as paraoxon and parathion. The OPH gene (opd) has been expressed in many hosts such as bacteria, insects, insect cells, fungi and Streptomyces spp. High level and soluble expression and correct folding of each protein are important factors. Fusion proteins including TRX, Gb1 and MBP are commonly used to increase solubility, improved folding and in some cases, stability. The present study evaluated OPH expression level, solubility proper folding and stability by cloning the opd gene into pET32a and pET21a and expressing the resulting vectors in E. coli shuffle t7. The pET32a vector encodes a fusion protein containing TRX that is not present in the pET21a. The results reveal increased expression level, moderate solubility, proper folding and stability in produced OPH by the pET32a-opd construct compared to the pET21a vector due to the presence of the fusion with TRX in pET32a vector

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Genetic variation in coconut black headed caterpillar (Opisina arenosella Walker) population in India

Chalapathi Rao N. B. V., Sabana A. A., Rachana K. E. and Rajesh M. K.

Opisina arenosella Walker or leaf eating caterpillar or the black headed caterpillar, a serious defoliator of coconut palm, is an outbreak pest in Andhra Pradesh. In order to characterize the genetic relationship among the O. arenosella collected from various districts of Andhra Pradesh, 625 bp of mitochondrial cytochrome oxidase subunit I (COXI) gene was sequenced for 18 larval samples collected from five districts of Andhra Pradesh. The sequence analysis data revealed that out of 625 sites, 14 sites (2.24 %) were variable (polymorphic) without any insertion or deletion and remaining 611 sites were invariable (monomorphic). Phylogenetic analysis revealed the existence of high genetic diversity among all samples collected from different districts. Eleven haplotypes could be observed among the 18 individuals. The high haplotype diversity and low nucleotide diversity support the high level of genetic diversity among the O. arenosella populations.

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Probing the mitochondrial genomes of Phytophthora spp. for the presence of mature miRNAs

Rahul C.U. and Rajesh M.K.

MicroRNAs (miRNAs) serve as gene regulators in a multitude of biological processes. The study of the differential expression of miRNAs present in any microorganism upon infection of its host could help in better understanding the process as well as provide a methodology to possibly mitigate the functioning of that particular entity. Phytophthora is an oomycete that belongs to the Stramenophile kingdom which is a major pest of plants worldwide. With more than 80 species identified worldwide, Phytophthora has the ability to attack a variety of crops. miRNA are a class of small non-coding RNA that regulate gene expression by the degradation of RNA or inhibiting protein translation. They play a major role in many biological processes which can influence an organism both positively and negatively. In this communication, we aim to identify any potential miRNAs that may be present in the mitochondrial genome of various species of Phytophthora. Disruption of the mitochondrial processes (which helps in maintaining cell homeostasis) of this pathogen, by manipulating mitochondrial miRNA molecules, could serve as a potential deterrent to the spread of this disease.

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A new expression vector for production of recombinant proteins in plant cells

Nguyen Tuong-Van, Nguyen Chi-Mai, Le Thi Thanh, Le Quynh-Lien, Tran My-Linh, Dao Thi Sen, Tran Thanh-Thu, Geert Angenon and Chu Hoang-Ha

The use of plant cells and plant virus based vectors to boost the expression of recombinant proteins is attracted for research recently. However, there are concerns that constant strong expression of foreign protein driven by plant virus genes may affect the viability of plant cells. Therefore, in this study, a modified tobacco mosaic virus (TMV)-based vector was developed to elevate the production of recombinant in plant cell suspension culture without inducing negative effects. At first cDNA of TMV was amplified from pTMV30B by double PCRs to eliminate coat protein gene (CP) and insertion of the specific recombination sites. The PCR product was then transferred to the gateway vector pCB-GW containing Arabidopsis thaliana HSP 18.2 promoter to form the expression vector pCB-HSP:TMV-GFP. Analysis of transgenic BY-2 cells revealed that under regulation of HSP promoter and the help of TMV, the level expression of GFP was significant improved in a short time. The maximum amount of GFP was obtained 3 day post 370C treatment. A further study will be done in a larger scale to confirm the benefit the modified expression vector for rapid, cost-effective, large scale production of recombinant proteins in medicine and industry.

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Determination of Total Gossypol and Free Gossypol Content in different varieties of Bt and Non Bt Cotton seed extracts by High- Performance Liquid Chromatography (HPLC)

Karishma R., Lakshmi Sahithya U., Suneetha P., Chinnababu Naik V. and Krishna M.S.R.

Gossypol exits in two different forms, free form and total form in cotton seeds. However, both the forms of gossypol can be toxic to animals which limit the usefulness of cottonseed as animal feed. The present investigation deals with estimation of total and free gossypol content from different Bt and non-Bt cotton varieties. Gossypol content of various seeds extracts of Bt and non-Bt of cotton varieties was conducted through an improved HPLC. Among the all Non-Bt varieties, JKCH 2245 had shown higher amount of total gossypol 2068 mg/kg and free gossypol of 4140mg/kg and Bt variety RCH-779 BG-II had shown lower amount of total gossypol 919 mg/kg and free gossypol 1814 mg/kg. Non-Bt varieties had shown less gossypol content and these accessions were advised for farmers to enhance their export demands. This can be helpful to the cotton seed oil industries in India for exporting gossypol free cotton seed oil.

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Standardization of an ELISA using Recombinant Nucleocapsid Protein to quantify antibody response against Newcastle Disease Virus in Chickens

Logeswari S., John Kirubaharan J., Rajkumar R. and Rajendran D.

The coding sequence of NP gene was amplified by RT-PCR, cloned and expressed in prokaryotic expression system-E. coli. The crude extract of expressed protein was purified, extensively dialyzed and further purified by expanded bed adsorption chromatography. Three indirect ELISA using recombinant NP were standardized and the antibody titres against NDV in serum samples were found to correlate among these three ELISAs. The correlation coefficient values were 0.3 to 0.6 which were higher than table values for correlation coefficient at 1% level. The empirical ROC values were observed to be 0.929, 1.0 and 1.0 for comparisons between RP-ELISA versusHI, RP-ELISA versus WVP-ELISA and R P-ELISA versus PEPELISA respectively which are more than the standard value of 0.8.

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Optimization of culture conditions and squalene enrichment from heterotrophic marine microalga Schizochytrium mangrovei PQ6 for squalene production

Anh Hoang ThiLan, Ha Nguyen Cam, Thom Le Thi and Hong Dang Diem

Squalene is a triterpenoid hydrocarbon and a strong antioxidant used extensively in the food, pharmaceutical and cosmetics industries. The objective of this study is to explore the optimal conditions for squalene production and the enrichment of extracted squalene from the biomass of Schizochytrium mangrovei PQ6 - a heterotrophic marine microalga isolated in Vietnam. The effects of temperature, yeast extract, initial glucose concentrations, culture age and treatment with terbinafine (an inhibitor of squalene monooxygenase in the sterol biosynthetic pathway) on squalene content and yield in the PQ6 strain were studied. The highest squalene content and yield reached 61.76 mg/g dry cell weight and 629.94 mg/L respectively at 3% glucose, 1% yeast extract concentrations and 28oC after 5 days of cultivation. The addition of terbinafine led to a slight inhibition of cell growth; in contrast, a noticeable increase in squalene content was observed at terbinafine concentration of 100 µg/L, corresponding to an increase of 56.44% in squalene content compared to control (without terbinafine treated). Squalene content extracted from the biomass of PQ6 strain increased 1.7 times through enrichment process via organic solvents partitioning. The obtained results suggest that there is tremendous potential in the exploitation of squalene from S. mangrovei PQ6 biomass.

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A Tabulated Review on Biological Applications of Semicarbazones, Thiosemicarbazones, Phenylthiosemicarbazones and their Transitional Metal Complexes

Suvarapu Lakshmi Narayana and Baek Sung-Ok

In the last few years, much interest has been directed towards the use of chelating ligands containing sulphur and nitrogen in analytical studies as well as in structural studies of metal complexes. The wide application and rapid growth in the popularity of sulphur ligands are due to their remarkable properties as potential donors to form stable as well as characterized complexes in which the back bonding from the metal ion is possible under favorable conditions. In the present paper we reviewed the biological applications such as anti-tumor and anti-microbial activities of semicarbazones, phenylthio- semicarbazones and their metal complexes reported since 2012.

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