Research Journal of Biotechnology

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Research Journal of Biotechnology

An adenovirus vector-mediated RNA interference inhibits efficiently rat stim1 expression in vascular smooth muscle cells and rat carotid artery

Liu Bei, Zhang Bin and Guo Rui-Wei

RNA interference (RNAi) is a general mechanism to knockdown a target gene expression but the efficient delivery of siRNA into cells is important for successful application of RNAi. In this study, we demonstrated an efficient and specific knockdown of stim1 (an important protein required for calcium influx and cells proliferation) in vascular smooth muscle cells and rat carotid artery which indicated a promising application of this adenovirus system in vascular physiological functions. Two target sequences for stim1 were chemically synthesized and recombinant adenoviruses for RNAi-mediated stim1 were produced. Rat aortic VSMCs were primarily cultured and transfected by an adenoviral vectors. Angioplasty of the rat left carotid artery was performed by using a balloon embolectomy catheter and transfected by an adenoviral vectors. Stim1 protein level was measured by western blot. An adenovirus vector for stim1 knockdown was successfully constructed. At 1, 3 and 5 day after infection with Ad-si/stim1 at MOI of 10 pfu/cells, the protein levels of stim1 was decreased significantly with compared to the Ad-empty group. The infection of Ad-si/stim1 virus (3 day after infection at 10 puf/cells of MOI) resulted in a robust decrease in store-operated Ca2+ entry (SOCE). Ad-mediated siRNA infected the rat carotid artery efficiently and inhibit the expression of stim1 significantly. This study develops a model system to study the function of stim1 in vivo and in vitro and the siRNA expression and specific gene knockdown can be achieved by adenoviral vector in vivo and in vitro.

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Molecular Characterization of Allelic Variants of Phytoene Synthase Gene (Psy) For Yellow Pigment in Indian Durum Wheat Genotypes

Sheoran Sonia, Singh Virender, Narwal Sneh, Dubey Neeraj and Gupta R. K.

Phytoene synthase (Psy), a critical enzyme in the carotenoid biosynthetic pathway, demonstrated high association with the yellow pigment (YP) content in wheat grain. In the present investigation, allelic variations in the Phytoene synthase gene were assessed in 76 Indian durum wheat genotypes including advanced lines from different agro-climatic zones of India using the functional markers developed from the sequence polymorphisms of Psy-A1 gene on chromosome 7A and Psy-B1 gene on chromosome 7B and to discuss the relationship between allelic variants of these Phytoene synthase genes with either low or high YP content to evaluate the feasibility of markers for selecting wheat cultivars. The results showed that with co-dominant marker YP7A, the frequencies of two alleles in 76 Indian durum wheat genotypes were 47.36% (Psy-A1a) and 39.47% (Psy-A1b). Three alleles Psy-B1a, Psy-B1b and Psy-B1e at Psy-B1 locus were found in Indian wheat genotypes. The frequencies of these alleles were 59.21%, 26.31% and 11.84% respectively. Data indicated the prevalence of Psy-A1a and Psy-B1a alleles in selected Indian genotypes. Psy-B1c and Psy-B1d were not detected in any of the selected Indian durum wheat genotype. The results showed that the functional marker YP7A and YP7B-2 were significantly correlated to the grain yellow pigment content. Phytoene synthase gene was amplified from seven Indian durum wheat genotypes and their molecular diversity was characterized. Use of molecular markers associated with YP content has the potential to improve selection efficiency for high or low YP content.

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Comparison of the multi gene regions for selection of potential barcode for Bipolaris spp

Sonavane Priti, Devi Prameela and Jagannath Raju

Bipolaris with Bipolaris maydis as type species is well known pathogen causing serious diseases i.e. Brown spot of rice [B. oryzae (Breda de Haan) Shoemaker], Brown stripe of sugarcane [B.stenospila (Drechsler) Shoemaker] and Southern leaf blight [B. maydis (Nisikado and Miyake) Shoemaker] of maize. Identification of organism is very difficult both by morphology and molecular. There have been no much studies to identify potential gene regions to group different Bipolaris species. In present investigation, 24 Bipolaris isolates belonging to five different species were used for multi regions (ITS, tef-1, β-tubulin, LSU and SSU) phylogenetic analysis to know the potential region to cluster the same species. ITS was found to be the best in species discrimination followed by tef-1and β-tubulin. LSU and SSU were unable to segregate Bipolaris species.

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Cloning and Characterization of Gene related to Gender CmACS-3 Gene in melon (Cucumis Melo L.)

Huijun Zhang and Feishi Luan

Being an organ of multiplication, flower is a vital constituent part of plant. About 90% angiosperms are perfect flower (bisexual flower) while the other parts are dioecism and monoecious. The Cucumis melon.L has a complicated vegetal pole. After we got the homologous fragment, a gene order of CmACS-3 had been gotten from the colon of monoecious melon which got 98% RSAC with the nucleotide sequence of CmACS-3. The length of CmACS-3 is 1865bp, and this sequence got a length of 1446bp with 481 amino acids. This gene shows up in the root, stem and leaf of melon. It has established a foundation of melon sexual differentiation.

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Polymorphisms at exons 3 and 4 of the growth hormone gene in Najdi and Naeimisheep of Saudi Arabia

Mahmoud A.H., Saleh A. A., Alagaili N.A., Alanazi. K M., Shafey T.M. and Abouheif M.A.

Growth hormone (GH) is a promising candidate gene marker for selecting production traits in livestock animals. The objective of the present study was to determine the polymorphism in exon 3, intron 3 and exon 4 of GH gene in 39 Najdi and 38 Naeimi sheep breeds of Saudi Arabia. A 934 bp fragment was amplified, sequenced and seven single nucleotide polymorphisms (SNPs) were identified: two synonymous in positions G985T and A1048G of exon 3, one non-synonymous in position G1386A of exon 4 and four in positions A1100G, C1103T, G1191A and C1289G of intron 3. Frequencies of allele G at positions G985T, A1048G, G1191A and C1289G were similar (P>0.05) between Najdi and Naeimi sheep, whereas its frequency at position A1100G was higher (0.16 vs. 0.02) in Naeimi (P<0.05) than Najdi sheep. At loci C1103T, G1191A and G1386A, alleles T and A were not found in the Najdi sheep and their frequencies averaged 0.07 in Naeimi sheep. Five genotypes in Najdi and eleven genotypes in Naeimi sheep were recognized at the seven polymorphic positions: three genotypes (G01-G03) in Najdi and five genotypes (G01-G05) in Naeimi were homozygous. The most common genotypes present in the Saudi Arabian sheep were genotypes G01, G02 and G07 with frequencies of 30.8, 43.6 and 15.4% in Najdi and 28.9, 23.7 and 18.4% in Naeimi sheep, respectively. Genotype G08 was found only in the Najdi sheep whereas genotypes G04-G06 and G09-G12 were detected only in the Naeimi sheep. Twelve representative genotype sequences from Najdi and Naeimi sheep have been deposited in the NCBI GenBank database.

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Detection of Stolbur Phytoplasma in Tomato by a specific DNA hybridization

Farag Azza G. and Mohamed Eman A. H.

The presence of stolbur phytoplasma in many crops all over Egypt is increasing severely. This pathogen was broadly observed in tomato leaves in a field located at Alexandria. Effective and accurate techniques depend on the PCR analysis with M1/P8 primer pair that amplifies a stolbur-specific, non- ribosomal fragments of a 720 bp. Therefore, we strongly recommend the application of DNA hybridization method for rapid and accurate detection of such economic pathogen.

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Production of intracellular β galactosidase using a novel Kluyveromyces marxianus DIV13-247 isolated from an Algerian dairy product

Boudjema Khaled, Fazouane-Naimi Fethia, Güven Kemal, Bekler Fatma Matpan, Acer Omer and Hellal Amina

β galactosidase (EC is an enzyme that cleaves lactose into glucose and galactose for different applications. It is extensively used to reduce the lactose intolerance people and to produce galacto-oligosaccharides (GOS) which are known as prebiotic food ingredient. In addition, it has been used to limitate the environmental problem caused by whey. In the present study, a novel yeast strain producing β galactosidase was isolated and further identified by physiological, biochemical and molecular methods. Then, some environmental and nutritional conditions for lactase production were studied. Based on the results of biochemical characteristics and the sequencing of internal transcripted spacer (ITS) rDNA gene, our yeast isolate belonged to Kluyveromyces genus and it was designated as Kluyveromyces marxianus DIV13-247. Moreover, the optimal culture conditions for yeast growth and β galactosidase production using this strain were found to be respectively incubation period of 18 hours, pH 5 and temperature of 30°C. It was also noted that lactose and urea were considered as appropriate carbon and nitrogen sources for best enzyme synthesis. The lactose hydrolysis using crude β galactosidase on both whey and lactose solution (5%w/v) was investigated. Its rate achieved 68.8% and 87, 4% respectively after 8 hours of incubation at pH 7 and temperature of 30°C.

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Quantification of bioactive components from medicinal herb Ganoderma lucidum using HPTLC and GC-MS techniques

Mohan Kannan, Padmanaban Muthusamy and Uthayakumar Venkatachalam

Ganoderma lucidum is currently popular medicinal herb and used in the formulation of nutraceuticals and as functional foods. In this study the bioactive components of G.lucidum were investigated using HPTLC and GC-MS techniques. HPTLC and GC-MS studies were carried out by Harborne12 and Wagner et al13 methods. Different compositions of the mobile phase for HPTLC and GC-MS analysis were tested in order to obtain high resolution and reproductive peaks. The ethanol extract of G.lucidum displayed the presence of 9 different types of flavonoids with 9 different Rf values ranging from 0.05 to 0.93. The results of HPTLC analysis of terpenoids demonstrated the presence of 12 different types of terpenoids with 12 different Rf values ranging from 0.05 to 0.94. The result of alkaloids demonstrated the presence of 11 different types of alkaloids with 11 different Rf values ranging from 0.01 to 0.92. GC-MS analysis on the bioactive components of ethanol extract resulted in the identification of twenty components for G.lucidum. Based on the result it can be concluded that the ethanol extract of G. lucidum is the potential natural resource for pharmacology and functional foods. The development of such fingerprinting from the mushroom G. lucidum is useful in differentiating the species from the adulterant and can act as biochemical markers in the pharmaceutical industry and systematic studies.

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Molecular identification and genetic variation of toxigenic and atoxigenic Fusarium verticillioides and its toxin-contaminated maize grains

Mahmoud Mohamed A. and Abd-El-Aziz Abeer R. M.

Nineteen isolates of Fusarium verticillioides were collected from sixty maize (Zea mays L.) samples from different markets located in Riyadh region, Saudi Arabia. The isolates were identified as F. verticillioides by its morphology and assured by the internal transcript spacer analysis (ITS1, ITS2 and 5.8S rDNA regions). F. verticillioides isolates were assayed by high-performance liquid chromatography (HPLC) for their ability to produce Fumonisin B1 (FB1), moniliformin (MON) and zearalenone (ZEA). Toxigenic F. verticillioides isolates (63.2%) were more prevalent than non-toxigenic isolates (36.8%). ISSR markers were used to characterize F. verticillioides isolates in terms of their genetic variability and their mycotoxigenic profiles. The intraspecific genetic similarity between F. verticillioides indicated a high level of genetic variation. Additionally, clustering of ISSR genotype was not related to toxigenic profiles of isolates.

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Preliminary test of functionalized ZnO2 against Bipolaris sorokiniana and other seed associated mycoflora for better wheat germination

Singh Nahar, Khandual Ansuman, Gupta Prabhat K. and Vaish S.S.

Bipolaris sorokiniana that causes foliar blight of wheat is one of the most serious worries for growers in warmer and humid areas around the world. Use of ecologically sound molecules for management of plant diseases is considered as one of the best options to achieve sustainability of ecology, agriculture and human health. Attempts were made to synthesize PVP functionalized ZnO2 nanomaterials to explore antifungal ability and impact on plant growth. The growth and spore germination of B. sorokiniana was significantly reduced. The seed germination was improved with good development of plumule and radicles. Moreover, the proliferation of wheat seed associated pathogenic and saprophytic mycoflora was completely inhibited. These new informations suggest use of PVP functionalized ZnO2 nanoparticles as an alternative to harmful fungicides for seed treatment and lower agricultural cost and environmental risk.

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High-yield expression and purification of recombinant antibody-alkaline phosphatase bifunctional protein against clenbuterol in Escherichia coli

Liu Xixia, Li Meiying and Wang Hong

Despite the commercial benefits of clenbuterol (CBL), its use as a feed additive in animal production is illegal. Therefore, rapid detection methods such as enzyme-linked immunosorbent assay (ELISA) are necessary to effectively control the quality of animal products. Herein, a recombinant single-chain Fv-alkaline phosphatase bifunctional protein against CBL (CBLscFv-AP) was prepared by optimizing the ion concentration of the medium. Results indicated a yield corresponding to 8.52% of the total bacterial protein. The bifunctional protein weighing 73 kDa was obtained at a yield of as high as 19.33 mg/L of culture after affinity purification, and its purity was as high as 98%, as revealed by reverse-phase high-pressure liquid chromatography. Direct competitive ELISA also showed that the purified CBLscFv-AP protein was bifunctional given that it demonstrates both CBL-recognized specificity and AP bioactivity. These results indicate that expression in Escherichia coli is an efficient and economical method for large-scale production of CBLscFv-AP bifunctional protein against CBL for downstream applications.

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QSAR Study on the toxicities of alcohols and phenols based on minimal redundancy maximal relevance and distance correlation feature selection methods

Deng Xiaolong, Tan Siqiao, Chen Yuan and Yuan Zheming

Toxicity prediction can provide important information for environmental protection. The toxicity predictions of 228 alcohols and phenols were performed by quantitative structure-activity relationship (QSAR). Feature selection can reduce the training time of modelling, improve the prediction accuracy and enhance the interpretability of a model. Both dependent variables (toxicity) and independent variables (molecular descriptors) of the QSAR data sets are usually continuous variables. The well-known feature selection method, minimal redundancy maximal relevance (mRMR) can eliminate redundancy and extract relevant features effectively but can only be applied to discrete dependent variables. The distance correlation (dCor) can detect the nonlinear correlation of two continuous variables. In the present work, a new mRMR-dCor feature selection method was developed by combining mRMR with dCor and used to construct the QSAR models for three datasets based on the retained molecular descriptors and support vector regression (SVR). mRMR-dCor feature selection method showed better predication performance (the Q2 of three datasets are 0.954, 0.941 and 0.981 respectively) than the reference feature selection methods and other methods reported in literature. In all, mRMR-dCor feature selection has a promising application prospect in the numerous domains of high dimensional feature selections such as QSAR.

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Non-thermal plasmas (NTPs) for inactivation of viruses in abiotic environment

Puligundla Pradeep and Mok Chulkyoon

Despite several successful disinfection strategies, process of controlling the environmental survival and transmission of human pathogenic viruses is becoming increasingly more difficult because of their emerging resistance to disinfectants. Therefore, range of non-chemical methods of inactivation is being explored extensively as an alternative. Especially, non-thermal, chemical-free techniques find wide application in the inactivation of air-borne, water and food-borne and surface-borne viruses. Among such methods, the application of non-thermal plasmas (NTPs) for viral inactivation is a relatively new technique and is quite promising. The present review evaluates comprehensively the studies of virucidal effect of NTPs against human pathogenic viruses in abiotic environment.

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