Research Journal of Biotechnology

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Research Journal of Biotechnology

Expression Analysis of HSP 101 Gene in Bread Wheat (Triticum aestivum L.Em.Thell.)

Singal Manjri, Yashveer Shikha, Singh Vikram and Dhillon Santosh

The present study was carried out on four genotypes of wheat WH730, WH 1021, WH 147 and WH 711 developed at CCS Haryana Agricultural University, Hisar. These four genotypes were raised in the field under normal and late sown conditions during 2012-2013. Data on physio- morphological traits like canopy temperature depression, grain yield, 100 seed weight and heat susceptibility index was recorded. The affect was more in varieties WH 147 and WH 711 as compared to WH 730 and WH 1021. The order of thermo-tolerance based on heat susceptibility index (HSI) was: WH 730 (0.42) > WH 1021 (0.68) > WH 711 (1.19) > WH 147 (1.53). Expression analysis of HSP101 (heat shock proteins) transcripts (HSP101, HSP101b and HSP101c) was also studied for the genotypes under controlled conditions where artificial heat stress (at 31ºC, 34ºC, 37ºC and 40ºC) was given to plants at the seedling stage. All heat shock transcripts (HSP101, 101b and 101c) were found to be expressed at all heat shock treatments and their expression was found to be maximum at 40ºC. WH730 showed the maximum level of gene expression of all the heat shock transcripts (HSP101, 101b and 101c) and WH 147 showed minimum level of gene expression. WH 730 showed maximum thermo-tolerance while WH 147 was thermo sensitive based on physio-morphological traits and gene expression analysis studies.

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Avian Shell – less culture model: A flexible model to study the events from stem cells to organogenesis

Rajendran Saranya, Kumar Pavitra, Rajendran Krithika and Chatterjee Suvro

Embryonic models are essential to study developmental and genomic features of an animal to develop therapeutic procedures for diseases in a wide spectrum of fields including cardiovascular development. However, embryonic models among higher vertebrates are not explored to study early development such as vasculogenesis and the development of therapeutics. Present study demonstrates that the avian shell-less culture model serves as a potential experimental model in the field of cardiovascular development. Here, we introduce a model that enables real time tracking of morphological alterations during physical phenomena including blood islands to plexus formation in an ex-vivo culture container starting from stage HH8 to HH27 with an embryo survival rate of 40%. Heart looping, a highly regulated and dynamic process could be studied with the proposed model. Further we have established the model to study gene expression pattern during developmental phase; specifically during vasculogenesis. Our model might become an inevitable choice among the developmental biologists to set up their experiment, analyze their images and acquire knowledge about the biologically significant gene expression during cardiovascular development. Thus, this model system has the potential to enhance our knowledge on developmental and transcriptomic analysis.

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Conserved miRNA detection in the ESTs of Ganoderma lucidum

Rahul C.U. and Rajesh M.K.

Ganoderma lucidum is a potentially deadly fungal pathogen belonging to the family Ganodermataceae that affects palms all over the world. Spread through spores, its mode of infection generally starts from the roots and is characterized by the presence of fungal basiocarps on the infected tree. Due to the huge financial losses inflicted upon the farming community, there is a need to find innovative measures to inhibit the spread of this destructive biotroph. miRNA induced silencing is a relatively new technology that is gaining traction in the field of plant pathogen inhibition. In this study, we aim to identify conserved miRNAs through bioinformatics tools present in the ESTs of G. lucidum. A total of 17 pre-miRNA hairpin loop structures were identified in the genome formed by a total of 9 unique miRNAs. Target annotation revealed that almost all of these miRNAs could have a part to play in propagating the spread of this pathogen. Thus, these miRNAs could be potentially upregulated or downregulated in a pathogen to restrict its parasitic functions.

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Biochemical characterization of insecticide resistance in geographic populations of Amrasca biguttula biguttula (Ishida)

Vimala, Bheemanna M., Chowdary Rajesh and Reddy Srinivasa Ronda

In the current study susceptibility of the twelve geographic populations of leaf hopper, Amrasca biguttula biguttula Ishida tothiomethoxam 25 WG was assessed using bioassays as well asenzymatic evaluations. Calculated LC50 values against nymphstages among different locations were highest in population of Nanded (0.41 ppm) followed by Coimbatore (0.40 ppm) and Raichur (0.38 ppm) compared to rest of the populations collected from different geographic population. These findings were further supported by biochemical characterization of insecticide resistance by determination of esterase activity for the populations. Esterase activity of individuals of Nanded population to 1-NA (Napthyl Acetate) and2-NA (Napthyl Acetate) was 0.19 and 0.56 μM product/min/mg protein respectively followed by esterase activity of Raichur population to 1-NA and 2-NA as 0.21 and 0.41 μM product/min/mg protein respectively. Highest AChE (Acetylcholinesterase) activity of enzyme was noticed in the population of Raichur (0.141) followed by Nanded (0.136) U/min/mg protein whereas wide range of GST activity was noticed in different geographic populations (58.78 to 527.78 pmol/min/mg protein). Analysis of variance showed that there were significant differences in esterase activities between the populations.

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Unraveling the soil bacterial diversity of Chloroxylon spp. from degraded soils of Uttar Pradesh

Upadhyay Neha, Sharma Shivesh, Rani Radha, Devi Shikha and Tiwari Ashish

Rhizosphere is the most vigorous and metabolically active section of the soil ecosystem. It is highly populated with a number of microbes directly or indirectly involved in the enhancement of plant growth. The bacterial species associated with the rhizosphere having plant growth characteristics are categorized as plant growth promoting rhizobacteria (PGPR). PGPR is most often used for promoting plant growth either by directly supplying nutrient availability to plant or by indirectly suppressing the growth of plant pathogen. In the present study, twelve bacterial isolates were isolated from the rhizospheric and bulk soil samples and screened for different plant growth promoting characteristics in vitro. The different parameters assessed were IAA production, phosphate solubilization, free N2 fixation, ACC deaminase activity and siderophore production. Rhizospheric effect was also analyzed by calculating the R: S ratio for different samples. R: S ratio was observed in the range of 1.70 to 2.90 highlighting the absence of antagonism in soil. Out of twelve isolates, five isolates were positive for IAA production and phosphate solubilization where as three were positive for siderophore production. Maximum IAA production and phosphate solubilization was recorded by NU75. Isolate NU75 was further screened for quantitative estimation of phosphate solubilization and it was evident that 73 µg/ml of phosphate was solubilized after 5 days of incubation. The most promising isolates were then selected and characterized biochemically. The growth promoting potential of promising isolates will be further used for development and promotion of plant growth in pots under green house conditions.

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Isolation and characterization of endoglucanases produced by microbes residing in the gut of Coptotermes curvignathus termite

Hii Hung Ling Monica, King Jie Hung Patricia, Ong Kian Huat, Bong Choon Fah Joseph and Mahadi Nor Muhammad

Bacteria and enzymes in the gut of termites play an important role to digest lignocellulosic material. Coptotermes curvignathus is one of the very few destructive species that can infest living plants. In this study, five bacteria isolated from C. curvignathus gut; four aerobic Bacillus spp. and an anaerobic uncultured bacterium were identified to produce endoglucanase with molecular size of 11 kDa which is significantly smaller than the endoglucanase produced by Reticulitermes speratus. Biolog reader identification showed that TG117 and N45/1 were Bacillus cereus/thuringiensis, TG111 was Bacillus pseudomycoides and TG005 was Bacillus mycoides. Endoglucanase produced by aerobic isolate NA45/1 showed promising potential as an industrial enzyme with significantly higher enzymtic activity than the commercial cellulase from Aspergillus niger (C1184 Sigma). Endoglucanase NA45/1 displayed enzymatic activity 0.3961 U at pH 9 and 45°C. The endoglucanase TG111 acted optimally at alkaline condition with 0.2294 U whereas endoglucanase TG117 functioned best at slightly acidic condition. This study showed that the termite gut has a wide range of endoglucanase enzymes with various optimum temperatures and pH.

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Development of linkage map and identification of QTLs responsible for fusarium wilt resistance in castor (Ricinus communis L.)

Tomar Rukam S., Parakhia M. V., Thakkar J. R., Rathod V. M., Padhiyar S. M., Thummar V. D., Dalal H., Kothari V.V., Kheni J. V., Dhingani R. M. and Golakiya B. A.

Fusarium oxysporum f. sp. ricini is a soil-borne fungal that causes wilt in castor which causes yield loss up to the extent of 85%. Through the screening of about 35 different genotypes of castor for fusarium wilt in the sick plot, two genotypes namely RG 2800 and JC-18 were identified as the most resistant and susceptible genotype respectively. The mapping population of 190 F2 was generated using the selected parents. The parental lines were screened against 786 primers set (520 RAPD, 100 ISSR and 166 SSR) and about 141 polymorphic markers were identified. The mapping population of 190 F2 was screened against pre-identified polymorphic markers for the development of linkage map and the same population was phenotyped for disease reaction on the scale of 1-5. The linkage map and QTL's were identified using onemap and R/QTL package of R software. The linkage map developed by using onemap software resulted in ten linkage groups with total map length of 1,551cM. The map length of individual linkage group ranged from a minimum of 78.1 cM to a maximum 261.3 cM. QTL on linkage group 6 and a putative QTL on linkage group 8 at threshold value of 90% were identified to be responsible for resistance against the wilt in castor. However at threshold value of 95%, only one QTL at linkage group 6 was identified with a LOD score of 13.5. Confidence interval at linkage group 6 indicated QTL location between 12-16 cM nearer to marker CST 73 and R 83. The results are of major importance for understanding the molecular background of wilt resistance in castor. These QTLs identified for wilt resistance have potential use in marker-assisted selection.

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The static extraction of lipid from microalgae Desmodesmus sp. MCC34

Nagappan Senthil and Verma Sanjay Kumar

The commonly used techniques for extraction of lipids from dry or wet biomass involve energy intensive steps such as cell lysis, high temperature and cell mixing causing substantial energy burden on the process. In present work, we report our finding on using a static method of mixing standard solvent with dry algal biomass without stirring. This extraction procedure was found to depend on the ratio of solvent volume to the biomass (SBR) and surface area factor (SAF). The kinetic study suggests that the static extraction followed Patricelli model of bi-phasic lipid extraction, consisting of a rapid washing step followed by the diffusion step. The results also suggest that the rate of lipid extraction in static process at optimum SBR and SAF, matched the rate of extraction obtained when lysed biomass was used or in the case where biomass was mixed (stirred) with solvent.

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Study on a straw degrading microbial complex DSG-3 and its application performance

Jiangwei Zhu and Yan Zhao

A microbial complex DSG-3 capable of degrading rice straw was bred from soil. It possessed excellent viability and could decompose rice straw effectively. The microbial complex DSG-3 has also been developed into a microbial preparation successfully, and the degrading enzyme activities of new microbial preparation during straw degradation were very high, its degradation capability was on the same level of the commercial preparation N1. In addition, the storage stability of new microbial preparation was well and could be stored in usual room temperature. Therefore, the microbial complex DSG-3 preparation was considered as a potential candidate for commercial application.

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Characterization of the Polysaccharides from Low-Quality Fresh Ginseng through High-Pressure Homogenization Process

Lee Hyeon Yong

The aim of this work was to obtain the polysaccharides having good biological activities from low-quality fresh ginseng by employing a simple high-pressure homogenization process with very high shear stress of 4,900,000 (1/sec). The optimal condition of this process was to obtain 2100x103 (g/mol) molecular weight of a crude polysaccharide. This polysaccharide was found to be composed of two polysaccharide fractions: F1, 856 x103 (g/mol) and F2, 1137 x103 (g/mol) respectively and interestingly enough, the fraction 2, F2 had high contents of uronic acid. This fraction was also found to have high biological activity, compared to the F1. The F2 polysaccharide showed high suppression of NO production. These results strongly indicate that high-pressure homogenization can efficiently destroy the crystal structure of intact ginseng polysaccharides as well as to break down the hard cell walls of low quality fresh ginseng, resulting in yielding the polysaccharides.

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Purification and Characterization of a Polyphenol Oxidase from Cimin Grape (Vitis vinifera spp., Cimin)

Faiz Özlem

A polyphenol oxidase (PPO) from Cimin grape was purified 11.2-fold by using a Sepharose 4B-L-tyrosine-p-aminobenzoic acid affinity column. The optimum pH and temperature of PPO purified from Cimin grape were 7.5 and 30°C respectively. The apparent Km was 19.5 mM and Vmax was 2378U/ mg protein. Ascorbic acid and sodium metabisulfite were potential inhibitors. Enzyme stability was higher than for most PPOs purified from other sources, with 90% of original activity retained after incubation for 2 hours at 80 °C. The activity of the enzyme increased by 145.7±2.0%, 138.7±2.4% and 130.5±1.4% in the presence of 1 mM Ca2+, Ni2+ and Co2+ respectively. This study gives essential information about Cimin grape PPO, an enzyme affecting the shelf-life of the fruit.

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Purification and characterization of Pleurotus ostreatus oxido-reductive laccase produced through solid state fermentation using Musa cavendish as a solid support

Nagarajan Balaji and Kannaiyan Sathish Kumar

The laccase enzyme was produced through solid state fermentation by employing the biomass of Pleurotus ostreatus supported onto the peel of Musa cavendish. The enzyme was produced under controlled environmental conditions viz. various pH, temperature and moisture content for achieving maximum activity. The crude laccase was purified by multi-stage purification protocol employing ammonium sulphate precipitation, dialysis and DEAE-sephacryl column gel permeation chromatography. The final purified laccase solution exhibited a purification fold of 3.76 with a specific activity of 4.40 U/mg. The final purified enzyme had a molecular weight of 66 kDa, thereby confirming the presence of the laccase enzyme when subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).

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Quantitative Trait Loci Mapping for Flowering and Yield-related Traits with a Recombinant Inbred Line Population of Maize (Zea mays L.)

Sa Kyu Jin, Park Kyong-Cheul, Ramekar Rahul Vasudeo, Park Jong Yeol and Lee Ju Kyong

In this study, a total of 80 F7:8 recombinant inbred lines (RIL), derived from a cross between dent corn inbred line, Mo17 and waxy inbred line KW7, were evaluated for 11 flowering and yield-related traits. Correlation analysis was performed among the 11 flowering and yield-related traits. DA showed the highest correlation with DS (0.747**). DS via ASI (0.619**), TC via SC (0.595**), CB via ER (0.535**) showed relatively high correlation coefficients than the other trait combinations. Quantitative trait loci (QTL) and epistatic interactions were detected for 11 flowering and yield-related traits. Our study detected a total of 32 QTLs and 40 epistatic interactions for 11 flowering and yield-related traits. The 15 QTLs were major QTLs which had more than 15% phenotypic variation (16.29~82.45%). qKT9 had the highest phenotypic variance (82.45%) in marker interval between umc1634 and wx1. The qDS1a and qASI1a were co-located within a region flanked by two adjacent SSR markers on chromosome 1 (umc1354 and bnlg1179) and qDS1b and qASI1b were co-located within a region flanked by two adjacent SSR markers on chromosome 1 (umc1166 and umc1976). In addition, two QTLs, qCB3 and qDA3 shared umc1949 and Two QTL, q10V5 and qDS5 also shared umc2373. Another QTL, qER10 and qASI10 shared umc1645. This tight link supported high correlation of each trait. Finally, these SSR marker pairs are useful selection tools for screening flowering and yield related traits. To perform MAS efficiently, it is important to identify genes or traits on the chromosomal segment linked to the target gene. Thus, the detection and confirmation of loci associated with main agronomic traits presented may provide greater opportunities for maize breeders to improve quality with marker associated selection.

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Enhanced Anti-proliferative and Apoptosis effect of Bortezomib-loaded Therapeutic System against Non-Small Cell Lung Cancer Cells

Wu Hui-Juan, Wu Hong-Bo, Zhao Yan-Qiu, Chen Li-Juan and Zou Hong-Zhi

In the present study, we have prepared a highly biocompatible and biodegradable cross-linked gelatin nanoparticle to increase the therapeutic efficacy of bortezomib to treat lung cancer. The formulation was characterized in terms of particle size, TEM, SEM andrelease characteristics. Biological characterization was performed in lung cancer cells. The formulated gelatin nanoparticles were nanosized with spherical outfit making it suitable for cancer applications. Furthermore, GNPB showed a sustained release pattern for BZB in phosphate buffer conditions. GNPB showed excellent and enhanced anticancer activity than free BZB by decreasing the cellular viability. Moreover, prepared formulations showed enhanced caspase-3 activity in NCI-H460 NSCLC cell line displaying its prominent apoptosis potential. Therefore, data from this study prove the antitumor potential of BZB by a nanodrug delivery system with gelatin as drug carriers. In this respect, bortezomib-loaded gelatin nanoparticles system would be a promising and potential therapeutic agent for the treatment of lung cancer.

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