Research Journal of Biotechnology

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Research Journal of Biotechnology

Selection of reference genes for quantitative RT-PCR studies in Uncaria rhynchophylla

Qianqian Guo* and Xiaojun Ma*

Page No: 1-6

Abstract: The real-time reverse transcription polymerase chain reaction (qRT-PCR) is a method for quantitative analysis and widely used for assessment of gene expression and regulation in medicinal plants such as uncaria (Uncaria rhynchophylla). During the practice, it is realized that selection of a reference gene has a great influence on data analysis.

In this study, we identified the four candidate reference genes (GAPDH, α-tubulin, NDUFV and β-actin) by analyzing the transcriptome and expression profiles from U. rhynchophylla and evaluated the accuracy of these genes as a reference gene in qRT-PCR by Delta Ct, Bestkeeper, Normfinder and geNorm algorithms. The results showed that the performance of GAPDH and β-actin was better than the other two genes while α-tubulin was the worst in the performance. Based on this study, we propose that GAPDH and β-actin are the most reliable reference genes for the normalization of qRT-PCR data in quantitative analysis.

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Defensive responses in Tea (Camellia sinensis) against Tea mosquito bug infestation

Suganthi M.*, Arvinth S. and Senthilkumar P.*

Page No: 7-11

Abstract: Induced resistance is one of the essential components of host plant resistance to insect pests. In this study induced defensive responses in tea with different levels of resistance to the sap sucking pest were studied. Enzymatic difference due to TMB (Tea Mosquito Bug) damage was studied and compared in susceptible and relatively tolerant tea cultivar to determine the markers for resistance and susceptibility.

The quantity of poly phenol oxidase (PPO), peroxidase (POX), ascorbate peroxidase (APX) enzyme activities has been enhanced in TMB tolerant tea plants. A decrease in superoxide dismutase (SOD), phenyl alanine ammonia lyase (PAL), catalase (CAT) enzyme activities is clear in TMB infested tolerant tea plants.

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Effects of Storage Conditions on the Stability of C-Phycocyanin in the Spirulina platensis Extract associated with Ultrasonic Process

Lee Hyeon Yong

Page No: 12-19

Abstract: The results of this work were to describe the long-term stability of c-Phycocyanin (C-PC) in the Spirulina platenssis extract obtained from an ultrasound process, compared with the change in C-PC content in phosphate buffer solution under the same conditions. At a low temperature of 4oC and in the absence of light, the C-PC amount in the extract (UE) and buffer solution (SS) decreased by less than 10% after one month of storage whereas approximately 40% of C-PC in both UE and SS was reduced at 40°C C in the absence of light compared with the initial contents of C-PC. It was also determined that the exposure to light significantly accelerated the decrease in C-PC contents which decreased to 60% of the initial content at 40ºC and even decreased to ca. 15% at 4ºC.

In general, storage temperature affected the stability of C-PC more than light. Interestingly, the C-PC in the extract was apparently more stable than in phosphate buffer at the same concentrations possibly due to the protection of light destruction by various components in the extract. The initial storage temperature was also observed to be important for a long-term storage since most C-PC in the solutions decreased within the first two weeks of storage. Long-term storage under harsh conditions also affected the structural changes of C-PC in the solutions by selectively breaking down α subunit out of C-PC, not β subunit, in absorption spectra analysis. These changes were also attributed to decrease in antioxidant activities of solutions, since C-PC was known to have strong antioxidant activities. Therefore, this study can provide useful information for maintaining an unstable C-PC in the Spirulina extract which is highly important in many industrial fields.

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Biochemical and Functional Properties of Proteins from common Legumes (Vigna mungo, Vigna radiata, Cajanus cajan and Cicer arietinum)

Hasan Anmar Nazar and Antony Usha*

Page No: 20-29

Abstract: Chemical composition and functional properties of proteins extracted from legumes of Black gram (Vigna mungo), Green gram (Vigna radiata), Red gram (Cajanus cajan) and Bengal gram (Cicer arietinum) were investigated. The results indicated crude protein contents of 23.2, 20.5, 22.8 and 18.9 % for Black gram, Red gram, Green gram and Bengal gram respectively. Green gram exhibited the highest carbohydrate content while Bengal gram showed the highest ash and moisture content. The dietary fibre (4.3%) and lipid content (2.3%) were found to be highest in Black gram. The minimum protein solubility of isolated proteins from all seeds was least at around pH of 3-4. High foam capacity was obtained in Green gram (saline extraction) and Bengal gram (water extraction). We observed high foam stability in Black gram (saline extraction) and Bengal gram (water extraction).

The highest oil absorption was found in Black gram while highest water absorption was found in Red gram. High emulsifying activity as well as stability in Green gram (saline extraction) and Red gram (water extraction) proteins were observed. Globulin and albumin (molecular weight ranges 14-66 kDa) were the major polypeptides in the protein isolate from all legumes. While Green gram contained one major polypeptide, more than two major polypeptides were observed in the proteins from other legumes.

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Biochemical isolation and characterization of Kojic acid derived from Aspergillus oryzae and Aspergillus flavus

Lakhawat Sudarshan, Kumar Vikram*, Choudhary Jignesh, Pathak Amrendra Nath and Kumar Shailesh

Page No: 30-35

Abstract: Kojic Acid (KA) is a fungal secondary metabolite which has several applications in pharma and cosmetic industries as potential skin whitening agent. In present study, kojic acid was produced from Aspergillus flavus and Aspergillus oryzae strains in three liters batch reactor. The HPLC, FTIR and XRD analysis confirmed the purity, average size and dimensions of kojic acid molecule. Unit cell dimensions noted were a= 5.518, b=8.8709 and c=5.0956. Actual angles of crystals are α==90° and β=91.66.

Kojic acid production was achieved up to 18.61g/l and 12.96 g/l for both A. flavus and A. oryzae respectively with double pH strategy. Aspergillus flavus is high kojic acid productive strain in comparison of Aspergillus oryzae. Moreover, double pH strategy significantly enhanced the kojic acid production in fermentations by both Aspergillus Species.

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Improved Cadmium Accumulation by Transgenic Tobacco Co-Expressing Metallothionein and Heavy Metal Transporter from Prosopis juliflora

Keeran Nisha S., Bansod Shama and Balasundaram Usha*

Page No: 36-41

Abstract: At a global level, the agricultural soil pollution with heavy metals is a serious ecological problem. To address this problem and remove soil contaminants, phytoremediation of heavy metals is a promising tool. Plants are engineered to overproduce various metal-binding and detoxifying proteins for enhancing their phytoremediation potential. In this study, cadmium (Cd) tolerance and accumulation were compared between control tobacco plants and PjHMT (heavy metal ATPase from Prosopis juliflora), PjMT1 (metallothionein from P. juliflora) or PjHMT PjMT1- (double construct) transformed tobacco plants. The results show that PjHMT-PjMT1-transformed plants accumulated higher amounts of Cd (11.3-fold) compared to control tobacco plants.

Furthermore, the heterologous expression of the same constructs in the yeast Saccharomyces cerevisiae (INVSc1) conferred greater Cd tolerance than control wild type yeast cells. This suggests that co-expressing heavy metal ATPase and metallothionein has a positive correlation with the phytoremediation potential of plants and could be adopted for Cd removal from contaminated soils.

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Effect of seaweed fertilizer on the growth, yield and nutrient quality of Lilium davidii var. unicolor and soil bacterial and fungal communities in a two-year field experiment

Yang Qiu, Zhong Kui Xie*, Ya Jun Wang and Ya Juan Zhang

Page No: 42-48

Abstract: A 2-year field experiment was conducted to study the effects of seaweed fertilizer at low and high levels on soil microbial community, plant height and chlorophyll content, the suppression of iron-deficiency chlorosis disease and the yield and nutrient quality of Lilium davidii var. unicolor. The results showed that the application of seaweed fertilizer significantly increased plant height and chlorophyll content, effectively controlled the iron-deficiency chlorosis disease, especially at a high level. The applications of seaweed fertilizer significantly enhanced yield and nutrient quality of Lilium davidii var. unicolor, even at a low application level. The maximum yield was achieved in the high-level seaweed fertilizer application that resulted in 26.62 % increase respectively compared to the control.

The addition of seaweed fertilizer significantly affected the composition of the bacterial community, enriching the quantities of beneficial fungi and reducing the fungal pathogens population. Thus, application of seaweed fertilizer could be a promising option for yield enhancement under Lilium davidii var. unicolor production.

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Persistent HSV and CMV viral load in periodontal pockets after non-surgical therapy

Pradeep Kumar Y., Ravishankar P.L., Anila E.N., Ahamed Suhail, Chakraborty Priyankar and Arunraj Rex*

Page No: 49-51

Abstract: The initiation of human periodontitis involves a web of complex interactions among Herpes viruses and specific pathogenic bacteria (Porphyromonas gingivalis) as well as tissue destructive inflammatory mediators (matrix metalloproteinase) and genetic factors (S34N). Various recent reports have revealed the presence of Herpes viruses in the periodontal pockets and highlighted its pathogenesis and the microfloral changes that occur due to its activation.

The main stay of periodontal non-surgical management is scaling and root planning which aims to remove the plaque and debris, thereby eliminates the pathogenic biofilm. Studies concerning the effect of non-surgical periodontal therapy on the viral load are scarce. Here we aimed to determine the effect of non-surgical periodontal therapy on the viral load in chronic periodontitis patients.

The present study aims to identify and compare the levels of Herpes Simplex Virus-1 and Cytomegalovirus in patients with chronic periodontitis before and after non-surgical therapy using quantitative real-time polymerase chain reaction (qRT-PCR). A total of 15 systematically healthy subjects with chronic periodontitis were screened. The clinical periodontal evaluation included measurement of the plaque index (PI-), periodontal pocket depth (PPD -) and clinical attachment level(CAL-). Subgingival plaque samples from > 6 mm periodontal pockets from the four deepest sites were taken before and after non-surgical –periodontal therapy. A qRT-PCR assay was used to identify genomic copies of periodontal HCMV and HSV-1. Data were analyzed by the Student unpaired t test and Pearson’s coefficient co-relation.

For the subgingival plaque samples taken from the patients with chronic periodontitis, the HSV-1 count was greater than CMV both before and after non-surgical therapy. PPD was significantly reduced after the non-surgical therapy, but there was no significant decrease in the viral load. Our quantification of HCMV and HSV-1 detected increased levels in the periodontal plaque samples of severe chronic periodontitis patients. Non-surgical periodontal therapy was not effective at reducing the viral load, thus highlighting the need for a more effective therapy.

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Plant mediated green synthesis of silver nanoparticles using seed extract of Psoralea corylifolia and to study its characterization and antimicrobial activity

Soni Manju A.* and Bhange Vivek P.

Page No: 52-59

Abstract:Green synthesis of nanoparticles has been an emerging research area now a day. In continuation of the efforts for synthesizing silver nanoparticles (AgNPs) by green chemistry route, here we report "green" route for the synthesis of AgNPs using seeds of Psoralea corylifolia (Bakuchi) which possess medicinal and anti-microbial properties. Here we have used a fast, convenient and environment friendly method for the synthesis of silver nanoparticles by biologically reducing AgNO3 with the aqueous extract of Psoralea corylifolia seeds.

The synthesized AgNPs have been characterized by UV-VIS spectroscopy, FT-IR, SEM and TEM analysis. These biologically synthesized AgNPs were also tested for antimicrobial activity against microbes Escherichia coli and Pseudomonas aeruginosa.

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Influence of Sporosarcina pasteurii SRMNP1 on the growth of plants under metal stress and its potential application in bioremediation of Cadmium and Copper polluted soil

Nivedhan Abilash, Reddy Gsumanth K., Ashfak Mohamed and Arul Jothi K.N.*

Page No: 60-66

Abstract: Heavy metal pollution in agricultural fields and water bodies due to bioleaching, chemical fertilizers, metal mining industries and other man-made activities poses a serious threat to agriculture and farming. Plants and microbes have been extensively used in the process of bioremediation to retain the fertility of farm fields. In this study, a combination of plants and bacteria were used for the bioremediation of cadmium and copper metal ions from the soil. The bioremediation efficiency of S.pastuerii SRMNP1 and its influence on plant growth was investigated using three different plants under induced, controlled environmental conditions.

The effect of S.pastuerii SRMNP1 on the growth of R. communis, S.melogena and V. unguiculata was elucidated using a pot experiment and the effect of S.pastuerii SRMNP1 on the bioremediation of copper and cadmium salts by the plants was determined by atomic absorption spectroscopy. AAS results confirmed the metal accumulation in S. melongena (Cu) and V. unguiculata (Cd and Cu) which were significantly higher than the metal accumulation rates in plants under control soil conditions. V. unguiculata accumulated 72% of Cu and 92% of Cd ions from the pot with artificial metal contamination without the bioinoculant whereas with the bioinoculant the accumulation was 67.5% and 67.1% of Cu and Cd ions respectively.

This study suggests that the bacteria and V. unguiculata or S. melongena can be effectively used in synergistic bioremediation applications in situ.

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Preparation and Characterization of Broad Specific Polyclonal Antibodies to Derivative of Sarafloxacin

Wei Dong, Lu Haibo, Fang Guozhen and Wang Shuo*

Page No: 67-74

Abstract: The antigen is the key factor in the preparation of antibody. In this study, Sarafloxacin(SALX) derivatives(SALX-NH2) was synthesized using Sarafloxacin and 3-Bromopropylamine hydrobromide. After purification by preparation of liquid phase, infrared spectroscopy(IR) scanner, electrospray ionization mass spectrometry(ESI-MS) and nuclear magnetic resonance(NMR) spectroscopy were used to identify the derivatives. SALX-NH2 was coupled with bovine serum albumin(BSA) and ovalbumin(OVA) to prepare the immune antigen SALX-NH2-BSA and coating antigen SALX-NH2-OVA, respectively. The complete antigen was identificated by ultraviolet spectrum. The Kunming mice were immunized with SALX-NH2-BSA to obtain the polyclonal antibody with high titer.

The optimum concentration of the coating antigen and the best dilution of antibody were determinated by chessboard method and the competitive indirect ELISA was established well detected SALX. The regression equation was y= -17.763x+89.239(R2= 0.9955); IC50 and LOD were 161.81 ng/mL and 18.62 ng/mL respectively. For all the competitive analogues, the assay has a high cross-reactivity to sarafloxacin (97.17%), norfloxacin (95.23%), ciprofloxacin (90.15%), enrofloxacin (88.38%), danofloxacin (27.35%), difloxacin (11.31%) and had no cross-reactivity to ampicillin, tetracycline and chloramphenicol. It indicated that the antibody with broad-specificity can be used to screen other several fluoroquinolones drug residues.

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Review on the Research carried out on in vitro Androgenesis of Peppers (Capsicum annuum L.) in Turkey

Nuray Çömlekçioğlu* and Şeküre Şebnem Ellialtıoğlu

Page No: 75-84

Abstract: Pepper is a one of most commonly consumed and economically important vegetable crop in the world. After China and Mexico, Turkey is the third biggest pepper producing country in the world. For that reason, it is important to develop new marketable cultivars by breeding programs. In vitro androgenesis methods are significant in plant breeding and they have been used effectively in rapid production of completely homozygous doubled haploid lines. In vitro production of haploids by androgenesis can be carried out using either anther cultures or isolated microspore cultures. In Turkey, the anther culture method is the most preferred and widely used method for obtaining haploid pepper plants.

This review provides information about research efforts in Turkey on pepper androgenesis and aims to summarize the experience gained about the factors influencing induction of androgenesis. As a common result of all the experiments carried out with the 167 different genotypes since the beginning (in 1983; Abak1), it was found that the genotype was the most significant and limiting factor on androgenic response. In addition to genotype, it was shown that composition of medium and additives, growth regulators, donor plant growing conditions, pretreatment of buds or anthers, developmental stage of microspores, age of the donor plant and incubation conditions were factors affecting androgenesis response in pepper.

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Plant microRNAs - Tiny yet significant regulators of disease resistance

Patwa Nisha and Basak Jolly*

Page No: 85-92

Abstract:MicroRNAs are 15-24 nucleotides long non-coding small RNAs found only in eukaryotic system. They are processed from a long transcript by RNA polymerase II and form into stem-loop primary miRNAs. Primary miRNAs ultimately form microRNAs by a Dicer-like enzyme. They regulate gene expression by loading into silencing complex and triggering their action either by cleavage or translational repression in the active cell machinery.

Although plant miRNAs have conserved role in plant development and biotic and abiotic stress resistance, yet there is a long way to go for effective use of miRNAs as a tool for the better growth and development of plants in this stressful world to meet the future challenges.

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