Research Journal of Biotechnology

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Research Journal of Biotechnology





Molecular cloning and expression of 3-ketoacyl-CoA synthase (3KeCs) gene in mulberry (Morus alba L.) under abiotic stresses

Dominic Kwame Kotoka and Zhao Weiguo

A cDNA sequence coding 3-ketoacyl-CoA synthase (3KeCs) gene (GeneBank accession number XM_010114628.1) was cloned from leaves of mulberry (Morus alba L.) based on mulberry expressed sequence tags (ESTs). Sequence analysis showed that its open reading frame (ORF) is 1413 bp encoding a protein of 470 amino acids with a predicted molecular weight and an isoelectric point (pI) of 53.42 kDa and 8.60 respectively. The 3-ketoacyl-CoA synthase had FAE1 (Type III polyketide synthase-like protein) domain with three active binding sites and belonged to condensing enzymes superfamily. A homologous analysis showed that 3KeCs gene was highly conserved in Morus alba and had high levels of similarity with other species like Morus Notabilis, Glycine max and Ipomoea nil. Phylogenetic analysis based 3-ketoacyl synthase gene from various species showed that Morus alba was closely related to Morus notabilis, Cucumis sativus, Juglans regia, Sesamum indicum and Ipomoea nil. The quantitative PCR analysis showed that the transcriptional level of 3KeCs mRNA had a significant change under drought, cold and salt stress conditions. The changes in the mRNA transcriptional levels of 3-ketoacyl-CoA gene in mulberry (Morus alba L.) show that the gene is responsive to abiotic stress conditions.

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Anticandidal activity of halophilic bacterium Vibrio azureus MML1960 isolated from Kelambakkam Saltpan, Tamil Nadu, India

- Abirami G., Ramprasath C., Arthi M. and Mathivanan N.

A halophilic bacterium was isolated from sediment samples of the Kelambakkam saltpan and it was identified as Vibrio azureus MML1960 by polyphasic taxonomy. The amplified sequence was submitted to the NCBI GenBank and the accession number is JN848577. Vibrio azureus MML1960 showed anticandidal activity against fluconazole-resistant Candida albicans. Optimization of the production medium with different pH, salinity and carbon and nitrogen sources was performed by the overlay method. The minimum inhibitory concentration (MIC) of crude extract was 0.375 mg/ml. GC mass spectra analysis was done to determine the compounds present in the crude extract. The active compounds were separated using silica gel column chromatography. The partially purified fraction 1 exhibited a maximum zone of inhibition (26mm) and the MIC was 0.0625 mg/ml against fluconazole-resistant Candida albicans.

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The pomegranate peel extract ameliorates inflammation via Akt/GSK-3β and NF-κB pathway in LPS-induced RAW 264.7 cells

Lai Hai-zhong, Chen Hua, Xin Xue-lei and Aisa Haji Akber

Pomegranate, as an astringent agent and antipyretic analgesic in China, has been used for thousands of years. The present study aimed at examining and explaining the anti-inflammatory effect and mechanism of the pomegranate peel extract (PGE) on lipopolysaccharide (LPS)-induced RAW 264.7 cells. PGE effectively decreased the production of pro-inflammatory mediators and cytokines, significantly decreased the expression of NO synthase (iNOS) and cyclooxygenase-2 (COX-2) in mRNA and protein level. We also revealed that activation of NF-κB was inhibited by PGE. Moreover, PGE did not only inhibit the degradation of IκBα and NF-κB activity but also the phosphorylation and activation of Akt, JNK and p38 MAPK. Thus, the present study demonstrated that PGE exerted the anti-inflammatory effects through the suppression of pro-inflammatory mediators and cytokine production in LPS-stimulated macrophages mediated by inhibition of NF-κB activation through Akt, JNK and p38 MAPK signaling pathways.

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DNA barcoding after cloning identifies taxonomically diverse botanical adulterants in the market samples of saffron

Varadharajan Bhooma and Madasamy Parani

Saffron (Crocus sativus L.), commonly used all over the world for culinary purposes is said to have many medicinal properties including antioxidant, anticancer, antidepressant, analgesic, diuretic and memory enhancing activities. Saffron is also consumed by pregnant women in many places. It is the world’s most expensive spice. Its high cost is due to the low production and the highly intensive labor required for its harvest. As a result, saffron is highly prone to adulteration. In our initial screening of commercial saffron samples by DNA barcoding, six samples were found to have been adulterated by mixing plant parts from more than one species. To identify the constituent adulterant plant species, we employed the DNA barcoding after cloning (DBAC) method using rbcL DNA barcode and TA cloning. The results showed the presence of at least four and up to ten taxonomically diverse plant species in each adulterated sample. Twenty nine adulterant species belonging to 18 genera and 10 families were identified. Some of the adulterant species were reported to be toxic to humans if consumed. Therefore, it is essential to identify the botanical adulterants in saffron to prevent fraudulent business practices as well as to enhance the safety of consumers.

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Bulk Segregant Analysis (BSA) for the improvement of Drought Resistance in Maize (Zea mays L.) Inbred Lines as revealed by SSR Molecular Markers

Farooqi Muhammad Qudrat Ullah, Ma Shi Jun and Lee Ju Kyong

In this study, maize inbred lines were used to evaluate performance under drought stress. Based on germination ability, 24 lines were screened out with tolerance or susceptibility to drought stress. Genetic diversity, relationship and population structure were evaluated by using 100 SSR molecular markers linked with drought tolerance in maize. A total of 465 alleles were identified with an average of 4.65 alleles per locus using bulk segregant analysis (BSA). Out of them, 199 were specific alleles with tolerance and 35 were susceptible to drought while the remaining 231 were shared between the two types. The major allele frequency varied from 0.39 to 0.61 with an average of 0.47. The gene diversity (GD) and polymorphic information content (PIC) average values from all lines were 0.64 and 0.59 respectively. Based on UPGMA analysis, four main cluster groups were identified as most of the drought tolerant lines were clearly discriminated from drought susceptible lines with 33% genetic similarity. On population structure analysis, 24 inbred lines with tolerance and susceptibility were divided into 3 groups. In addition to BSA, a total of 88 SSR’s were identified as specific to tolerance and 29 SSR markers were specific to susceptible lines. Among these, bnlg1627, umc1946, dupssr30b, bnlg1812 and dupssr24 SSR markers had great potential for the improvement of drought resistance in maize. Our results were in good agreement with previous studies for drought tolerance using SSR molecular marker and will be useful for further maize breeding studies.

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Role of sequentially extracted fractions of Costus pictus in inhibiting oxidative stress: in vitro study

Angel Steffy and Reddy Amala

Reactive Oxygen Species (ROS) are the lead reasons for an enormous number of degenerative diseases and dietary disorders. Natural Indian traditional medicine suggests that, naturally derived compounds are capable of protecting against free radicals. These could be lead active phytoconstituents of the plant. This had portrayed a great interest in research with natural anti-oxidants. So, an attempt has been made in order to review different in vitro models for the estimation of their anti-oxidant properties due to their exposure to natural products from Costus pictus. In vitro antioxidant efficacy of hexane, dichloromethane, ethyl acetate and methanolic extracts of Costus pictus has been tested through confirmatory assays viz. DPPH, Hydroxyl, reducing power and also TBARS. The methanolic extract of this leaf showed potential free radical scavenging activities, reducing ability and lipid peroxidation (LPO). This study indicates the free radical scavenging activity of Costus pictus which can be used for treating various free radical mediated ailments. Ascorbic acid has been used as standard for the study of antioxidant activity. Methanolic extract showed a higher degree of DPPH and hydroxyl radical scavenging activity, reducing powers and inhibition of lipid peroxidation which was about 93±1.87, 30.98±1.89, 87±1.33, 88±1.56 at 250 µg /ml respectively. The above results help us to substantially conclude that bioactive components are extracted well in high polar solvents which mainly include flavonoids and related polyphenols. These compounds present in Costus pictus may be active lead for potent antioxidant potency.

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Isolation, Medium Optimization and Identification of amylase producing strain from potato fields

Kaur Navneet and Singh Rattandeep

The enzymes produced from various micro-organisms are very much stable can be obtained easily. Amylases are the most widely used enzymes obtained from micro-organisms. In this experiment, studies upon the isolation and medium optimization of bacteria which produce amylase taken from potato fields have been undertaken. The bacteria which produces amylase was isolated from soil and isolated colonies were tested for starch iodine test. In this test zone of clearance were observed which confirmed the production of amylase by the isolated bacteria. The maximum amylase activity was found at temperature of 30-50ºC of temperature whereas the optimum pH was observed at pH 7 where it showed maximum amylase activity. Amylase activity was also checked for various carbon sources as well as for various nitrogen sources, among carbon sources highest amylase activity were found in case of glucose (1.8 U/ml) whereas in case of nitrogen sources yeast extract showed the highest amylase activity when incubated for 24 hrs. The effect of various concentrations of EDTA was also checked by incubating with different concentrations of EDTA where the amylase activities were found to increase only till 0.2% of EDTA. The 16S analysis have shown that the identified strain is lysinibacillus xylanilyticus.

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Computational and functional analysis of an alkaline serine kinase gene (aprN) from the genome of Bacillus subtilis NRRL B -14196

Kevin Kumar V., Rajnish K.N., Muthukumaran C. and Manojkumar N.

The incidence of cardiovascular disease has been increasing in recent years. Medications used for treatment of stroke include enzymes such as streptokinase and tissue plasminogen activators. Due to the high medication cost and their side effects, research has been aimed at the development of fibrinolytic enzymes from microbial sources. Most of the fibrinolytic enzymes are serine kinases and so far, Bacillus sp is the choices for microbial sources. The present study attempts to describe the bioinformatics and experimental validation of a Bacillus subtilis strain that produces alkaline serine kinase In this study, a combination of in silico analysis and experimental validation of an alkaline serine kinase from Bacillus subtilis NRRL B-14196 was performed. The results of bioinformatics analyses were validated using experimental and literature references. We have analysed the Bacillus subtilis NRRL B- 14196 genome which harbours a putative gene that is annotated as alkaline serine proteases. Conserved domain analysis predicted it to be a member of the serine protease family of peptidases. The predicted theoretical pI and aliphatic index values suggested AprN as a stable protein.

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Anti-proliferative effect of silver nanoparticles in HeLa cells due to enhanced oxidative stress

Kakade Datta P., Arora Sandeep and Ambwani Sonu

Silver nanoparticles (AgNPs) are one of the promising products in the nanotechnology industry. AgNPs have many potential biomedical applications viz. in the field of high sensitivity bimolecular detection and diagnostics, antimicrobials and therapeutics, biomedical optical imaging, biological implants (like heart valves) and many more. However, there is growing concern related to potential effect of AgNPs to the human/ animal health and environment. Considering the above fact, the study was undertaken to evaluate potential cytotoxic effect of silver (Ag) nanoparticle on human cervical cancer cell line (HeLa) and its possible mechanism. The cytotoxicity of AgNPs was investigated in HeLa cells through microscopic examination as well as MTT assay after exposing HeLa cells to various concentrations of AgNPs. AgNPs induced oxidative stress in HeLa cells was evaluated through Lipid peroxidation (LPO) and reduced glutathione (GSH) assays. Microscopic examination revealed alteration in cell morphology like increased cell granularity, clump formation, cell rounding and cell detachment in comparison to control untreated cells. MTT assay displayed that AgNPs significantly increased the cell mortality in a dose-dependent manner with maximum non-cytotoxic dose (MNCD) and minimum cytotoxic dose (MCD) dose of 10µg/ml and 20µg/ml respectively. Half lethal concentration (LC50) of AgNPs was determined to be 82µg/ml in HeLa cells. AgNPs were found to induce elevated levels of oxidative stress at MNCD and MCD both as indicated by significant increase in lipid peroxidation and depletion of glutathione (GSH) levels.

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Expression of structural gene of the bacteriocin nisin in mammalian cells

Sudhamani M.

In order to develop mastitis resistant cattle, genes of bacteriocin nisin operon need to be expressed in mammalian cell lines. Genes nisA, nisB and nisC were amplified from the genome of Lactococcus lactis subsp. lactis. The genes nisA and nisC were cloned in PCRTOPO3.1 vector. However, nisB could not be cloned. The genes nisA and nisC were sub cloned under the Cytomegalo virus (CMV) promoter in PCI vector. The linearized construct containing the nisin genes under CMV promoter was electroporated into CHO cell lines. Consequently, CHO cell lines integrated with nisA gene were obtained. CDNA analysis of the nisA integrated cell lines indicated that there was transcriptional expression of nisA.

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Optimization of expression conditions for code-optimized Arabidopsis protein phosphatase 2A catalytic subunit in Escherichia coli

Xixia Liu

This study describes the optimization of expression conditions for code-optimized Arabidopsis protein phosphatase 2A (PP2A) catalytic subunit in Escherichia coli. The code-optimized Arabidopsis PP2A catalytic subunit gene was cloned by PCR and sequentially inserted into the expression vector pET22b to express the Arabidopsis PP2A catalytic subunit in E. coli strain BL21(DE3)pLysS. SDS-PAGE and Western blot analyses revealed that the PP2A catalytic subunit is 36 kDa in molecular weight and is bound to anti-His monoclonal antibody. After the optimization of expression conditions, the expression yield increased from 17.43% to 20.06% and the recombinant plasmid did not affect the growth of the host bacteria. The present findings indicated that the Arabidopsis PP2A catalytic subunit can be produced in E. coli strain BL21(DE3)pLysS.

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The causal agent for leaf curl with necrosis in firecracker flower plants identified as Tobacco streak virus in Andhra Pradesh

Arundathi M., Manohar P., Radha A., Padma J.G. and Ramesh B.

Crossandra infundibuliformis (Firecracker flower) is one of the economically important flower crops in India and Sri Lanka. In this crop, leaf curling with necrosis was noticed on some plants during the field inspection at Dumpalagattu and Panyam villages of Kadapa and Kurnool districts of Andhra Pradesh respectively. To know the causal agent of the disease, total of 44 leaf samples from Dumpalagattu (12 samples) and Panyam (32 samples) showing leaf curl with necrosis were randomly collected and initially tested for Tobacco streak virus (TSV) by DAC- ELISA with TSV antiserum. Out of 12 samples of Dumpalagattu, 2 were positive for TSV and none of the samples of Panyam was found positive for TSV. The 2 positive samples were further confirmed by RT-PCR using TSV CP (coat protein) gene specific primers. The resulted PCR product of the virus of approximately 700 bp was subsequently sequenced and confirmed as TSV by NCBI-BLAST analysis. Sequence analysis of the present virus showed maximum identity of 99 % and above with TSV isolates of onion (Tamil Nadu), groundnut (Anantapur), okra (Maharashtra), sunn hemp (Delhi), mung bean (Delhi), onion (Tirupati), green gram (Tirupati), jasmine (Kadapa), jasmine (Kurnool), groundnut (Kadiri), soya bean (Tamil Nadu) and guizotra (Jalna) at both nucleotide and amino acid levels.

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Gene Expression Pattern of Metallothionein 1 and 2 in response to in vitro Regeneration of Canola (Brassica napus L.) Cultivars

Abdelmigid Hala M., AlMalki Faizah A. and Darwish Hadeer Y.A.

A comparative study of gene expression patterns during different phases of in vitro regeneration of six canola cultivars was conducted in this research. Calli were obtained from shoot meristem explants and grown on MS (Murashige and Skoog) medium, supplemented with auxin and cytokinin for callus initiation, control plants were propagated from seeds. Expression profiling of two metallothionein genes (MTs); namely BnMT1 and BnMT2 at different phases of tissue culture was analyzed using semiquantitative reverse transcription PCR (sqRT-PCR). The results revealed the superior transcripts of BnMT1 over BnMT2 in control plants within all studied canola cultivars. Approximately, all cultivars exhibited dramatic upregulation of BnMT2 in in vitro tissue culture propagated plants at different phases of culture, compared to control plants. On the contrary, low expression of BnMT1 could be observed in both studied phases of calli initiation. When the six cultivars were compared, BnMT1 transcripts were relatively equal in all cultivars whereas a remarkable differential expression in BnMT2 was recorded according to the origin and pedigree/selection history of canola cultivars. In case of in vitro regenerated plants, responsive variability appeared within cultivars in both BnMT1 and BnMT2 expression profiles. The results showed confirmed correlation between regeneration potential and gene expression patterns of both BnMT1-2 in the studied canola cultivars. Differential expression of MTs genes was exhibited by some cultivars suggesting genotypic differences sensitivity of canola cultivars to in vitro regeneration. These genes also, could therefore be useful as molecular markers for somaclonal variation, particularly in canola.

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PGPR: A Boon in Stress Tolerance and Bio control

Maxton A., Singh P., Aruna A., Prasad S.M. and Masih S.A.

Due to climate changes rainfall has become more erratic resulting in reasonable reduction in crop yield. Plants exposed to water deficit or saline soil can result in either the reduction of crop productivity or abandonment of many agricultural areas. Toxic heavy metal pollutants of industrial wastes and agricultural chemicals pose deleterious effects on plant growth and human health by entering the food chain. Under stress conditions, plant biosynthesizes higher levels of plant hormone ethylene using 1-aminocyclopropane-1-carboxylic acid (ACC) as a precursor. This stress ethylene inhibits the elongation of plant root, shoot and suppress leaf expansion. ACC-deaminase producing rhizobacteria can reduce stress by producing surface exopolysaccharides that bind to cations including sodium (Na+) and develop soil sheaths around the plant root which reduces influx of Na+ into the stele restricting Na+ influx in roots. Rhizobacteria are capable of producing IAA and GBA that increase water uptake capacity as a result of root proliferation. These rhizobacteria have developed different heavy metal tolerance mechanisms involving biosorption, exclusion, bioaccumulation or precipitation both in intracellular and external spaces.

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