Research Journal of Biotechnology

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Evolutionary analysis and structural insights in Brain type Cytochrome P450/Aromatase of Stinging catfish Heteropneustes fossilis

Sundaray Jitendra Kumar, Rasal Kiran Dashrath, Swain Pranati and Jayasankar Pallipuram

The brain type cytochrome p450 is the enzyme playing vital role in sexual differentiation as well as secondary sexual character development. To date, most of the studies on aromatase have focused on the expression pattern, promoter activity and function in sexual differentiation. As the computational algorithms/tools are being developed, they have revolutionized the way of study for in-depth analysis using existing nucleotide/protein sequence data. There is no evidence addressed for phylogenetic analysis for investigating conservation and 3D structure of aromatase protein in the Stinging catfish. The stinging catfish, Heteropneustes fossilis is one of the important species in aquaculture sector. The evolutionary analysis was performed using Neighbor-joining and UPGMA methods using CLC genomic 7.5.1 workbench tool followed by bootstrap test (100 replicates) performed to validate the phylogenetic tree/cladogram. We have generated 3D structure of aromatase protein of stinging catfish using homology modelling with help of template 4KQ8 (54 % identity) using tool Modeller 9.1 which was further validated using SAVES server in Procheck and Verify3D. The Ramachandran plot depicted that 89.4% residues lies in favored region followed by 9.7% in additional as well as allowed region and 0.9 % residues found to be in outlier region. In order to understand global network of aromatase protein, we have used STRING 9.1 tool and speculated that this protein interacting with several other protein, strong association/interaction with 2 proteins such as estradiol 17 beta dehydrogenase3, 3-beta steroid –dehydrogenase-1 with 0.988 and 0.979 confidence score respectively. In addition to this, we also investigated structural and functional consequences of SNPs on structure of aromatase using algorithms like PANTHER, PROVEAN and I-Mutant 2.0. We revealed impact of SNPs on the protein structure and its function using sequence-based tools.

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HPLC analysis and Antioxidant activities of Holoptelea Integrifolia

Kavitha Alli and Narasu Mangamoori Lakshmi

There has been a renewed interest in naturally-occurring antioxidants from fruits, vegetables and plants. This study is aimed at identifying antioxidant compounds present in the extracts of the Holoptelea integrifolia. The antioxidant properties of methanol, acetone and ethyl acetate extracts of Holoptelea integrifolia were determined using the 2,2-DPPH method, FRAP method and reducing power assay. The total phenolic content was estimated through the use of the Folin-Ciocalteu method. The total flavonoid content was determined according to Kim et al7 method. Ascorbic acid, Gallic acid and Quercetin were used as standards for evaluating radical scavenging activity, total phenolic content (TPC) and total flavonoid content (TFC). Results derived from the 2,2-DPPH method, FRAP method and reducing power assay showed that the stem extracts of acetone had the highest antioxidant activity. The TPC and the TFC values also showed similar results. The HPLC analysis of the extracts also confirmed the trend derived from the assays. The HPLC analysis showed large quantities of Gallic acid and Rutinin plant extracts. Results showed that the stem extract of acetone and the leaf extracts from methanol had the highest antioxidant activity compared to other extracts.

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In vitro clonal propagation of Rauwolfia tetraphylla, a relative of Indian snakeroot plant

Rohela Gulab Khan, Bylla Prasad, Chithakari Ravi, Korra Rajender and Reuben Christopher

Rauwolfia tetraphylla L., an endangered medicinal plant and a relative of Indian snakeroot plant (R.serpentina) hold an important position in the Indian traditional system of medicine and has other immense applications. A protocol for in vitro clonal propagation of Rauwolfia tetraphylla was developed using nodal and shoot tip explants. Nodal and shoot tip explants cultured on MS medium supplemented with Kn (1.5 mgL-1) + TDZ (0.25 mgL-1); BAP (2.5 mgL-1) + TDZ (0.25 mgL-1) produced 11-16 axillary shoots after 4 weeks of culture. The shoots that proliferated from nodal and shoot tip explants on MS + BAP (1.5 mg/L) + TDZ (0.25 mg/L), when transferred to half strength MS basal medium produced maximum mean shoot length (cm) (5.43 ± 0.86). Similarly the shoots that proliferated from nodal and shoot tip explants on MS + BAP (2.0 mg/L) + TDZ (0.25 mg/L), when transferred to half and full strength MS basal media produced maximum mean shoot length (cm) (6.41 ± 0.34 & 7.05 ± 0.71). Axillary shoots developed roots (100 %) when cultured on MS medium supplemented with IAA (1.0 mgL-1) + IBA (1.0 mgL-1). The survival rate of the plantlets was 90.0 % in field conditions.

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Gene cloning, expression and functional characterization of a novel acyl carrier protein gene JcACP from Jatropha curcas

Fan Shi and Deng Ming-Hua

Acyl carrier protein (ACP) is a small acidic protein that acts as an essential cofactor in many biosynthetic pathways depending on acyl transfer reactions. In this study, a new full-length cDNA of JcACP was obtained by RT-PCR technique from the energy plant jatropha. The nucleotide sequence analysis of the JcACP gene revealed that jatropha JcACP gene encodes a protein of 137 amino acids that belongs to PP-binding superfamily. Sequence alignment showed that its deduced amino acid sequence had high similarity with other ACPs. QRT-PCR analysis showed that JcACP was expressed in different tissues including the seed, leaf, root, flower, stem and pericarp. During the seeds development, the JcACP gene displayed a bell-shaped pattern of expression which had peak expression at 50 DAF (day after fertilization). The results demonstrated that JcACP gene is required for lipid biosynthesis involved in both endosperm development and storage lipid accumulation.

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In vitro assessment of Phaseolus vulgaris L. lectins activities against various pathogenic and beneficial microbes‏

Nciri Nader, Ben Ismail Hanen, Ben Aissa-Fennira Fatma and Cho Namjun

The mechanism by which kidney bean lectin PHA (phytohemagglutinin) provokes dramatic changes of the small intestinal ecosystem upon feeding is not entirely clear yet. The objectives of this work were to screen in vitro the interaction of an extract of Twila beans variety grown in Tunisia with a panel of eleven microbes and to evaluate the effect of PHA on the growth behavior of Lactobacillus delbrueckki subsp. bulgaricus isolated from a commercial yogurt. The activity of the lectin was checked by hemagglutination test. The interaction between the microbial suspensions and the bean extract was assayed in test tubes incubated for 1 hour at room temperature and then left overnight at 20ºC. The kinetic behavior of L. bulgaricus strain cultivated in MRS Broth medium was assessed in the presence of PHA. Results indicated that bean extract agglutinated human as well as animal erythrocytes with a higher specificity for group blood cell O and sheep erythrocytes. Bacteriological binding assays showed that Twila extract failed to agglutinate all the tested microorganisms. PHA did not stimulate or inhibit the growth of L. bulgaricus. These observations suggest that lectins present in Twila beans, when ingested; might not have any biological effects towards pathogenic and beneficial microbes.

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Screening of potential PGPR candidates as future biofertilizers-A strategic approach from lab to field

Gupta Garima and Jha Prabhat Nath

A vast increase in population, urbanization and industrialization has shrunken agriculture land and thus, affected demand vs. food supply ratio. Exploitation and manipulation of beneficial bacteria-plant association can be most effective and eco-friendly to enhance plant growth and productivity in sustainable manner. Various bioinoculants tested in several crops are found to be effective under laboratory conditions but showed inconsistencies in field studies. Therefore, present study aimed to adopt a systematic approach from lab to natural conditions for providing better and stable bioinoculants. In addition, to broaden the application of selected bacterial inoculants, cross infection studies were performed. Ability of endophytic bacteria to promote growth of pearl millet plant was tested at laboratory, greenhouse and field condition. They were screened and sorted out for various levels on the basis of various plant growth parameters. Bioinoculants of field trial were inoculated to wheat plants to confirm their endophytic nature and cross-infection capability using species-specific PCR. Most of the bio-inoculants promoted plant growth better in sterilized than unsterilized soil. In microcosm studies, some of the isolates showed synergistic effect on plant growth. In field studies, PM10461 was the best and consistent inoculant followed by PM9404 and PM9426. Cross-infection capability and endophytic colonization of these strains in wheat broaden the application to other crops. In future studies, various formulations (consortia) which showed better plant growth promotion in microcosm studies, can be tested at field level and efficient inoculants can be converted into commercial biofertilizer for wide usage and industrial production.

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Zea mays NAC transcription factor family members: their genomic characteristics and relationship with drought stress

Liang Li, Yiwen Ma, Shihuang Zhang, Zhuanfang Hao and Xinhai Li

NAC (NAM, ATAF1/2 and CUC2) has been verified to regulate biological responses in various biotic and abiotic stress-resistant activities. In Zea mays, 190 NAC proteins downloaded from the Plant transcription factor (TF) database website were identified and given their unified names here. Of them, 177 maize NAC proteins that showed at least two conserved subdomains of NAC TF were used for phylogenetic analysis. The NAC proteins were compared to 18 known functional NAC TFs in Arabidopsis and rice and a total of 195 NAC proteins were phylogenetically classified into five groups. The NAC-IV group was predicted to be a SNAC (stress-responsive NAC) subfamily. For them, 13 SNAC transcripts in the SNAC subfamily showed a response to drought stress and nearly all of these transcripts in the roots under drought stress and eleven transcripts in the leaves under 12 h of drought stress were up-regulated. Most important, ZmNAC010312, ZmNAC080308 and ZmNAC030295 of SNAC subfamily were further validated to be drought-responsive candidate genes by differential expression analysis. These findings are important for drought-responsive candidate gene selection in all maize NAC genes and advances in the functional analysis of the NAC family in Zea mays.

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Propensities to ATP binding sites in Myosin II domains

Rastogi Gargi, Meenakshisundaram N. and Sankaranarayanan Kamatchi

The function of many proteins depends on their interaction with small molecules or ligands. ATP is one such important ligand that plays critical role as a coenzyme in the functionality of many proteins. There is a need to develop method for identifying ATP interacting residues in ATP binding proteins (ABPs) in order to understand mechanism of protein-ligands interaction. Myosins are group of proteins driven by ATP hydrolysis and are very essential in cellular activities. The conservation in the ATP binding residues in myosins can lead to better understanding of mutant states during diseases. In this study we have studied ATP conserved sequences in several isoforms of myosins. This study will thus pave way for targeting ATP sequences during drug delivery mechanisms.

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Effect of different combinations of growth hormones and its interaction on callogenesis

Al-Khateeb Abdullatif Ali and Al-Khateeb Suliman Ali

This paper reports the effect of different combinations of growth hormones on fresh and dry weights on callus cultures of two date palm (Phoenix dactylifera) cultivars Khalas and Sukary. MS-medium fortified with 2,4-D in combination with 2ip was the best for the production of callus fresh weight in cv. Khalas while MS-medium that contained NAA with 2ip was the best for callus growth in Sukary. In Khalas, MS-medium supplemented with 10mg/l NAA+1.5mg/l 2ip gave the highest(2.476gm) quantity of callus fresh weight compared with the other treatments while the lowest fresh weight was recorded in 15mg/l NAA + 0.5mg/l 2iP (1.17 gm) and 10 mg/l 2,4-D alone (1.28 gm). In Sukary, the MS-medium supplemented with 10mg/L2,4-D + 1mg/L 2ip gave the highest (1.6571g) callus fresh weight compared with all hormone treatments. However, this combination produced less callus fresh weight in cv. Khalas. On the contrary,10mg/LNAA + 1.5 mg/L 2ip gave the lowest fresh weight of 1.2088gm in Sukary, gave the highest callus fresh weight in Khalas.The callus dry weight followed a similar trend. In Khalas the media combination which gave the highest fresh weight, also produced the highest (0.1534 gm) dry weight. However, the cv Sukary responded differently and gave the highest callus dry weight of 0.2450 gm in media with 15 mg/L NAA + 0.5mg/L 2iP compared to cv Khalas which gave the lowest (0.0981gm) dry weigh under this combination. Correlation Coefficient also revealed genotypic differences both in callus fresh and dry weights. However, interactions were very weak in almost all the media combinations used.

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Isolation, screening and characterization of Bacillus subtilis NB3 isolate for phytase production

Gangoliya Shivraj Singh and Singh Nand Kumar

Phytase enzyme producing bacteria were screened and isolated from poultry soil in phytase specific medium (PSM). Among the 20 isolates, one of the best isolates NB3 has high phytase activity. This result showed to isolate NB3 produced 35 mm clear halo zone on solid PSM and 170 U/ml in phytase specific broth medium. Enzyme produced extra cellularly by NB3 isolate in exponential growth of late stages. The upper limit production of phytase by this isolates was achieved after 60 hrs incubation at 40oC at pH 5.5 and the best nitrogen and carbon sources for highest phytase production were optimized as yeast extract and glucose respectively. The isolated strain NB3 characterized by 16S rDNA sequencing and NB3 showed adjoining correlation with Bacillus subtilis due to phylogenetic study.

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Oral immunization against H5N1 using transgenic tobacco cells expressing a multiple-epitope peptide of HA and NA

Tuong-Van N., Chi-Mai N., Thanh L. T., Quynh-Lien L. and My-Linh T.

Plant-cell-suspension culture is one of plant-based recombinant protein production systems showing promise in producing oral vaccine for infection diseases such as avian influenza. This study is a part of research on the plant-based vaccine against highly pathogenic H5N1 avian influenza. We have previously reported a plant expression system based on TMV under transcriptional control of heat inducible promoter of protein HSP18.2. In this work, we applied our expression tool toward the development of plant cell-derived vaccine candidate against avian influenza virus. One hundred and ninety-eight amino acids of LTB and the viral HA and NA epitopes were replaced of GFP in pTMV HSP-GFP and the recombinant gene then was transformed into BY-2 tobacco cells through Agrobacterium-mediated method. The recombinant protein was expressed to high levels in BY-2 cells and was recognized by C-myc tag protein antibodies. The efficacy of the antigens in transgenic BY-2 cells was determined in mice and chickens. The increased production of specific antibodies was observed. This initial study demonstrates the feasibility of using plant virus-based vectors and plant cells for expression of antigenic epitopes of H5N1 to again avian influenza.

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Essential Oil Composition, Antimicrobial and Bioactive Properties of Origanuım hypericifolium, An Endemic Plant Species grown in Turkey

Hüseyin Fakir, Ahmet Abdullah Us, Mustafa Sagdic and Fatih Tornuk

In this study, Origanum hypericifolium that is an endemic species to Turkey was collected from Denizli (a South-Western province of Anatolia) and its essential oil was characterized for its antimicrobial and bioactive properties and volatile composition. p-Cymene that is the precursor of carvacrol was the most abundant constituent observed in the volatile composition while thymol, borneol, and γ-terpinene were the other major compounds. Agar diffusion method was used for determination of antibacterial activity against Escherichia coli O157H7, Salmonella enterica subsp. enterica serotype Typhimurium, Listeria monocytogenes and Staphylococcus aureus. E. coli O157:H7 was found to be the most susceptible species to the essential oil with the 15.5 mm inhibition zone. Bioactive properties of O. hypericifolium essential oil were much higher (P<0.05) than the hydrosol. Total phenolic content, DPPH scavenging activity and total antioxidant activity of the essential oil were measured as 104.928 mg GAE/g, 38.95% and 9.979 mg TA/mL respectively. In conclusion, this study highlighted that O. hypericifolium had a good potential with its high antimicrobial and bioactive properties to be used as a strong phytochemical agent in pharmaceutical and food industry. Therefore, its cultivation and production should be improved.

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Effect of Extraction Solvents on the Skin-Whitening Activity of Rhodobacter sphaeroides

Kim Nam Young and Lee Hyeon Yong

The skin-whitening activity of extracts from the photosynthetic bacteria Rhodobacter sphaeroides was compared based on the utilized extraction solvent including water, 100% or 70% ethanol, 100% or 70% methanol, hexane, or chloroform. Among those solvents, the 0.000078 wt% extract from 100% ethanol had the least cytotoxicity against human skin fibroblasts (93.4% viability) followed by 92.1%, 88.2%, 74.9%, 84.3% and 92.1% viability for the water, 70% EtOH, MeOH, 70% MeOH and hexane extracts respectively. The 100% ethanol extract also showed the highest tyrosinase inhibition (87.07%) compared to 75.01% and 72.93% for the hexane:EtOH (3:1) and 70% EtOH solvents respectively. Melanin synthesis was markedly inhibited (83.9% activity compared to control) by the addition of the 0.002 wt% extract from the 100% ethanol solvent. These results were confirmed by the gene expression levels of microphthalmia-associated transcription factor (MITF) and tyrosinase which were upregulated 13-fold and 6.92-fold respectively after the addition of bacteriochlorophyll. The 100% ethanol extract also showed the highest antioxidant activity, 68.9% DPPH free radical scavenging which implies that the 100% ethanol solvent more effectively extracted antioxidant substances than the other solvents and therefore resulted in greater skin-whitening activity. These results strongly indicate that 100% ethanol is the best solvent for extracting photosynthetic bacteria, even though 70% ethanol has been considered the most appropriate solvent for natural plant resources;100% ethanol could also be employed to extract other bioactive substances from bacteria and other microorganisms.

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Photosynthetic and antioxidant responses of Salicornia bigelovii Torr. to the salt stress

Zhang Bianjiang , Zhou Feng and Tang Ning

In this study, H2DCF-DA fluorescence of root and the response of the antioxidant and C4 photosynthetic enzymes to salt stress were determined in Salicornia Bigelovii Torr. In response to NaCl stress, PEPC and NADP-ME enzymes activities were induced. At the concentration NaCl stress (200-400 mmol∙L-1), the system of antioxidant enzymes could scavenge the reactive oxygen species in time. H2DCF-DA fluorescence was not remarkably observed in root region under 400 mmol∙L-1 but in control suggesting that the roots were less damaged. These results indicated that Salicornia Bigelovii possessed the high photosynthetic capacity and exhibited protection mechanism against medium salt stress (200-400 mmol∙L-1) by maintaining the specific activity of antioxidant enzymes.

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Effect of ACC-deaminase producing Bacillus cereus brm on the growth of Vigna radiata (Mung beans) under salinity stress

Kothari Vishal V. and Vyas Bharatkumar Rajiv Manuel

Bacillus cereus brm isolated from the rhizosphere of Brassica nigra growing in the saline desert of Radhanpur (India), was identified on the basis of biochemical and 16S rDNA analysis. Salinity stress induces higher levels of ethylene in plants which limits crop production. This increased concentration of ethylene can be reduced by using plant growth promoting rhizobacteria (PGPR) producing ACC-deaminase. Bacillus cereus brm produces ACC deaminase (0.22, siderophore (85% U), IAA (3.36 μ and solubilizes phosphate (150 μ B. cereus brm has the ability to grow over pH 5-11 and in the presence of up to15% NaCl. Talc-based B. cereus brm formulation (2×108 cfu.g-1) was prepared and evaluated for its plant growth promoting activity. Root elongation of Vigna radiata in salt stress conditions (1.2 S.m-1) was 3.6 cm while in control it was 2.5 cm. The results show that salinity stress decreased mung bean growth significantly but inoculation of B. cereus brm reduced the inhibitory influence of salt stress on mung bean growth.

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