Research Journal of Biotechnology

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Research Journal of Biotechnology

Molecular cloning and expression characterization of a novel JcFatA gene from Jatropha curcas

Wen Jin-fen, Huo Jin-long and Deng Ming-hua

In this study, a new full-length cDNA of JcFatA was obtained by RT-PCR technique from the energy plant Jatropha. The nucleotide sequence analysis revealed that Jatropha JcFatA gene encodes a protein of 369 amino acids that belongs to hot_dog superfamily. Sequence alignment showed that its deduced amino acid sequence had high similarity with other FatA. qPCR analysis showed that JcFatA was expressed in different tissues including the seed, leaf, root, flower, stem and pericarp. The JcFatA gene displayed a bell-shaped pattern of expression which had peak expression at 50 DAF. The results demonstrated that JcFatA genes are required for lipid biosynthesis involved in both endosperm development and storage lipid accumulation.

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Bioactive constituents, antioxidant and antimicrobial activities of three cultivars of wax apple (Syzygium samarangense L.) fruits

Moneruzzaman Khandaker M., Jahan Sarwar Md, Nashriyah Mat and Boyce A.N.

The present study represents report on bioactive constituents, antioxidant and antimicrobial activities of three cultivars of wax apple fruit. Results revealed that fruits of both Masam manis (pink) and Jambu madu (red) cultivars are the ultimate sources of anthocyanin, carotene and vitamin-C. On the contrary, chlorophyll and titratable acidity content were higher in fruits of Giant green (green) cultivar. The amount of trace elements was high in pink followed by red and green cultivars. The highest amounts of total phenols and flavonoids were in methanol extracts compared to ethanol fruit extracts and these polyphenolic compounds were correlated with antioxidant activities (R2 = 0.98). In addition, ethanol extracts of leaf, bark and fruit showed good antioxidant activity with IC50 values ranging from 283-358 μg mL-1 and antimicrobial activity as compared to methanol extracts. Among the cultivars, red cultivar performed the best antimicrobial activity. Bark and fruit extracts of green cultivar showed the highest antioxidant activity as measured by rabbit erythrocytes haemolysis. LC-MS/MS analysis showed several peaks with fragmentation patterns corresponding to the high antioxidant activity of the water extracted fraction. This current study has shown that wax apple cultivars are ultimate source of bioactive constituents, trace elements and metabolites that have the potential to serve as antioxidant and antibacterial compounds beneficial for human health and may serve as a lead to the development of new pharmaceutical products.

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Genetic diversity in cultivars and landraces of rice as revealed by SSR markers

Akhtar Sabina, Wani A. Shafiq, Bhat M. Ashraf, Wani Shabir and Wani Nawsheeba

Rice yields have now apparently reached a plateau possibly due to use of common dwarfing gene sources and alleles contributing yield potential that has led to narrowing genetic base of these high yielding cultivars. Therefore, the use of local germplasm calls for its evaluation and characterization before embarking on their use in a systematic breeding programme. In the present study 40 SSR primers generated a total of 250 alleles across these genotypes with a range of 2 - 11 alleles per locus and a mean of 6.89 alleles per locus. The PIC values, measuring allelic diversity and polymorphism ranged from 0.490 (RM2592) on chromosome-1 to 0.806 (RM585) on the chromosome-6, with a mean PIC value of 0.719 per locus. Similarity coefficients (measuring relatedness) were used to construct the dendogram and clustering pattern. Pairwise similarity coefficients ranged from 0.10 to 0.89, with the average similarity coefficient around 0.40. Similarity coefficient value revealed presence of good genetic diversity among these genotypes at the molecular level. Clustering pattern revealed grouping of these genotypes into 5 clusters. All the landraces got grouped into 2 clusters viz. cluster-I (13) and cluster-II (7) except Rehman Batt-1 which got grouped in the cluster-III which mostly contained genotypes for higher altitude conditions. High yielding varieties for valley basin conditions formed cluster-V (5 cultivars), while cluster-IV contained cutivars mostly of K-series.

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Evaluation of sample preparation protocols for proteomic analysis of Oenococcus oeni

Zhang Guoqiang

Proteomics has been introduced to the study of Oenococcus oeni but the protein preparation method is still unsatisfactory with respect to protein spot number, separation and resolution, and this has limited the application of a proteomic approach to the study of O. oeni. In this study, the protocol for protein isolation from cells of O. oeni cultivated in partially insoluble complex medium was investigated. The goal was to make the obtained extraction samples suitable for the two-dimensional electrophoresis, thus make the further proteome analysis possible. Compared to non-denatured procedure, the denatured one, urea-thiourea in the rehydration solution enhanced the re-solubilization of protein pellets but led to heavy horizontal streaking in the 2-DE gels. 20% TCA/acetone precipitation with phenol/chloroform/ isoamyl alcohol (25: 24: 1, v/v/v), efficiently eliminated the interference substances from the cell lysate. High protein loading amount improved the resolution of some low abundance proteins but did not adapt to the high abundance proteins. And it was also important to collect cells at appropriate culture time according to the analysis target. With the optimized protein extraction protocol, the protein expression patterns of O. oeni during different growth phase were analyzed.

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In vitro direct plant regeneration from mature cotyledons of Clitoria ternatea L.

Mishra Awadhesh Kumar, Pandey Krishna and Tiwari Kavindra Nath

An efficient in vitro regeneration protocol was established for direct shoot regeneration from mature cotyledon explants of Clitoria ternatea. Adventitious shoots were regenerated on Murashige and Skoog (MS) medium supplemented with various concentrations of 6 benzyl amino purine (BAP), kinetin (KIN) and thiadiazuron (TDZ). The highest frequency (93.76±0.14) and maximum number of shoot buds/explant (36.06±0.78) were obtained on MS medium supplemented with 3.0 mg/l BAP. The microshoots were elongated in GA3 (1mg/l) containing MS medium. The highest rooting (90%) and maximum number of roots (8.33±0.33) per shoot and root length (5.50±1.80cm) were obtained when shoots were pulse treated with 500 mg/l IBA (Indole 3 butyric acid) for 5 min. Complete plantlets were then hardened, acclimatized and transplanted to natural conditions where they exhibited 80% survivability.

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Identification of Thermophilic Strains from Geothermal Areas in Turkey by using Conventional and Molecular Techniques

Derya Yanmis, Mustafa Ozkan Baltaci, Medine Gulluce and Ahmet Adiguzel

A survey of isolation was practised by obtaining water samples from various thermal facilities in order to find out new types of thermophilic bacteria and to characterize them via phenotypic and genotypic methods. Hence, the test strains were first grouped according to their colonial morphologies and were characterized via conventional (morphological, physiological, biochemical) and molecular [Fatty Acid Methyl Ester profiles (FAME), 16S rRNA sequencing and BOX-PCR] methods. As the result of the research, it was determined that FAMEs method failed in the identification of thermophilic bacteria at the species level whereas BOX-PCR technique was quite successful in exhibiting genetic differences among thermal isolates. On the other hand, as the result of 16S rRNA sequence, 4 of the isolated strains (A7-A10) were determined to resemble Bacillus licheniformis at a rate of ≥97% while 2 of them (A1 and A13) resembled to Anoxybacillus kaynarcensis, 1 of them (A2) to A. flavithermus, 1 of them (A3) to A. rupiensis, 1 of them (A4) to A. thermarum, 1 of them (A5) to A. gonensis, 1 of them (A11) to B. thermoamylovorans,1 of them (A12) to Ureibacillus thermospaericus, 1 of them (A14) to Brevibacillus brevis, 1 of them (A6) to B. thermolactis and again 1 of them resembled to B. thermoruber (Z1) at the same rate. Their amylase and cellulase productions were also examined and analyzed and the enzyme producing isolates that were biotechnologically valuable were presented to be used in the prospective studies.

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Enhancement of Anticancer Activity of Low Quality Fresh Ginseng by Lactic Acid Fermentation and High Pressure Processing

Choi Woon Yong and Lee Hyeon Yong

Low-quality fresh ginseng was extracted at 240 bar for 20 min and then fermented by Lactobacillus rhamnosus HK-9 at 36°C for 48 h (LFH). At a concentration of 1.0 mg/mL, the LFH extracts had 28.43% and 21.78% lower cytotoxicity towards HEK293 cells and HEL299 cells respectively than the low-quality fresh ginseng and red ginseng extracts obtained from conventional 100°C water extraction. The LFH extract stimulated the highest human immune B and T cell proliferation, with cell counts of 9.33×104 cells/mL and 15.33×104 cells/mL respectively. An in vivo experiment using ICR mice showed that the variation in body weight in ICR mice treated with 100 mg/kg/day fresh LFH ginseng extract was considerably lower than that observed for the control and other groups. The survival time for the group treated with this extract was 35.65% longer than that of the control group. Additionally, this extract showed the highest tumor inhibition activity which was 70.64% for sarcoma-180 solid tumors and the specific secretion of IL-6 and TNF-α was increased by up to 35% in response to treatment of cells with the LFH extract. The results revealed that the stronger anticancer activities of the LFH could be caused by enhanced immune activation. It was also found that the high pressure extraction process followed by the fermentation selectively produced some of the ginsenosides that are known to exist only in red ginseng. These results indicate that the extracts from low quality ginseng could improve anticancer activities.

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Cloning, In-Silico Characterization and Homology Modeling of Phaseolin gene from Common Bean (Phaseolus Vulgaris)

Rosy Rani, Singh Jitender, Kumar Pankaj, Thakur Yogindar, Shukla Pradeep and Misra Pragati

Phaseolin is the major seed storage protein containing a conserved barrel domain. It comprises a complex set of glycoprotein heterogeneous in their polypeptide and belongs to cupin superfamily. In the present study the total genomic DNA was extracted from the cotyledons of Phaseolus vulgaris and phaseolin gene was amplified, cloned and sequenced. Multiple sequence alignments showed that Phaseolin proteins possess highly conserved DNA binding domains and extensively conserved motifs consisting of a 1263 bp of protein encoding DNA. The present study depicts a high quality model structure of phaseolin using SWISS- MODEL web server. The QMEAN4 score was evaluated for the generated models. The modeled residue ranges from 1 to 421 bp based on the template [2PHL_A] (2.20) with 98% of Sequence Identity and E-value: 0.0. Secondary structure with structural pattern from the protein sequences with helix, sheets and coils was obtained by Psired program. The quality testing of protein was performed using various qualities testing software’s. Energy minimization was performed by Chiron Rapid Energy Minimization server and the Ramachandran plot of designed model was calculated through rampage server. ProFunc web tool was used for the analysis of the structure obtained. The model was found to decode sequence information correctly in true functional state.

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Rapid Identification of Peanut Hybrids with SSR-PCR

Hong Yanbin, Liu Hong, Li Xingyu, Chen Xiaoping, Liang Xuanqiang and Huang Shangzhi

To fulfill high efficiency peanut hybrid identification, an SSR-PCR rapid detection system was established by simplifying the process of DNA template preparation. After that, true hybrids from six F1 populations were identified by morphological observation and SSR-PCR. The result showed that true and pseudo peanut hybrids were difficultly distinguished by morphological observation when hybrid parents were similar in morphology whereas it was easy by SSR-PCR. In the six F1 populations, 38-56% of putative hybrids were identified as true hybrids by SSR-PCR. Hybrid rates of peanut might vary among different combinations even in reciprocal crosses.

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Demonstration of anticancer effect by apoptosis exhibited by biotechnologically synthesized silver nanoparticle of Indigofera aspalathoids leaf extract

Krishnasamy L., Masilamaniselvam M. and Jayanthi K.

Flow cytometric analysis using Hep3B cells and western blotting were applied to study the protein expression of various cellular targets (such as Bcl-2, P53, Bax, caspase 3 and caspase 9) involved in apoptosis. The biotechnologically synthesized silver nanoparticle effectively proved to be an inducer of caspase 3 gene. Other changes in genes on Hep3B cells indicated changes in mitochondrial structure which are associated with programmed cell death (apoptosis). Our findings proved that the synthesized silver nanoparticle is a valuable new chemical entity in the discovery and development of apoptotic research related to anti-cancer therapy.

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The Error Grid Analysis of invasive and indigenously developed noninvasive technique based blood glucose readings obtained from the effect of various glucose concentration sample solutions over blood glucose levels on the human subjects

Chowdhury Koushik Md., Srivastava Anuj, Sharma Neeraj and Sharma Shiru

Noninvasive blood glucose monitoring technology with significant clinical accurate measurement capability should have a beneficial impact over medical care system for diabetes and its related risk managements. Moreover blood or other body fluid less technology should increase patient compliances as well as robust control over fluctuating blood glucose levels. Herein, we represent the noninvasive technology and evaluate its efficiency for detecting the respective glucose concentration induced blood glucose levels on the human subjects. The purpose of this experiment was to observe the effect of various glucose concentration sample solutions like 25gm/100ml, 50gm/100ml and 75gm/100ml over blood glucose levels on the fasting 10 human subjects during the time duration of 1hour and 2hour after respective glucose solution consumptions. Moreover, the invasive and indigenous noninvasive technology based blood glucose level readings correlations were compared and evaluated through Clarke and Parkes Error Grid analytical approaches. The experimental findings indicate that blood glucose levels increase with increase in concentration of glucose solutions in human subjects. At the same time, the blood glucose levels were regulated and controlled by the individuals’ body glucose homeostasis response mechanisms. Similarly the Clarke and Parkes Error Grid analysis of the obtained invasive and noninvasive blood glucose readings showed that the measurement readings occupy the medically significant A and B zones respectively. Experimental study indicates the impact of glucose concentration over blood glucose levels and potential use of indigenously developed noninvasive technology based on amplitude modulated ultrasound with infrared technology for noninvasive blood glucose level determinations.

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