Research Journal of Biotechnology

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Research Journal of Biotechnology

Molecular characterization and expression analysis of ring-box protein1 (Rbx1) gene in Citrus reticulata

Tang Wenwu and Wu Xiulan

Self-incompatibility (SI) causes seedlessness in citrus. Skp1-cull-F-box proteins play important role in SI reactions. Rbx1 is component of Skp1-cull-F-box. However, studies did not reveal role of Rbx1 gene in SI reaction of citrus. In this study, Rbx1 gene was cloned. Three bases differed in amino acid content and open read frame sequence between Wuzishatangju (WZSTJ) and Shatangju (STJ). After self-pollination, Rbx1 expression rapidly increased from 0 to 3 d h. Highest expression was detected in day 3 and then decreased within days 4–7. After cross-pollination, Rbx1 expression gene slowly increased from 0 to 4 days and declined from days 5 to 7. Expression level of self-pollination was six-fold than that of cross-pollination. Phylogenetic tree analysis showed that Rbx1 has close genetic relationship with Citrus sinensis (L.) Osbeck (Cs4g07360). Rbx1 proteins from WZSTJ and STJ were successfully extracted in prokaryotic cells. Pollen germination rate of WZSTJ was significantly inhibited by Rbx1 protein. At higher concentrations, inhibition effect was more apparent.

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Rapid detection of the flic-d gene of Salmonella typhi using loop-mediated isothermal amplification

- Pasupathi Rathinasabapathi

Typhoid fever caused by Salmonella typhi remains a major health threat in many countries. In this study, a rapid, sensitive molecular detection method for Salmonella typhi was developed by Loop-mediated isothermal amplification (LAMP) of fliC-d, flagellin-encoding gene. A set of four specific primers were designed targeting six distinct regions of the fliC-d gene. The target DNA was amplified as early as 45 mins of incubation at 65°C in a simple water bath. The sensitivity of this method was found to be one femtogram of genomic DNA which is equivalent to 1.93 genomic copies and it is 1000 times more than that of PCR. The amplified products were identified by direct visualization of green fluorescence under ultraviolet light with the addition of Calcein in the reaction. This direct visualization method could be used to develop LAMP based on-field detection kit for Salmonella typhi.

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Cytokinin ameliorates the abiotic stress severity in Chinese Scholartree (Sophora Japonica L.) through regulation of chlorophyll fluorescence, antioxidative response and proline metabolism

Nguyen Van Quy, Zhou Qian Yi and Zhao Zhong

Cytokinins (6BA) are acknowledged for regulating plant growth through adverse environmental constrains such as drought and salinity, which particularly limit plant growth and therefore, affect plant senescent. This study was conducted to evaluate 6BA treatment (20 mM) in both drought (50% dryness) and salinity stress (50 mM NaCl) having Chinese Scholartree (Sophora japonica L.) as test specimen. The result showed that drought and salinity both decreased total chlorophyll content significantly, however 6BA treatment ameliorated the adverse effect of these constrains. The phenomenon was moreover, clarified based on the enzymatic activities such as Superoxide dismutase (SOD) and Catalase (CAT) where it was observed that both the stress conditions imposed an oxidative stress causing a downfall of these enzymes. However, the treatment of 6BA regulated these enzymatic activities and lowered the effect of stress on the treated plants. In relation to enzymatic activities, oxidative biomarkers, particularly the Superoxide radical (O2-) as well as the Malondialdehyde content (MDA) and proline content increased stress conditions indicating a significant oxidative stress. Interestingly, the cytokinin treatment regulated these physiological activities indicating its significant role in ameliorating the stress effects on the subject plants. Furthermore, from current findings, it is revealed that 6BA treatments can stimulate the bud to sprouting of S. japonica seedlings.

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Anti-biofilm efficacy of curcuma aromatic- a traditional herb against coronary stent infection causing bacterial strains

Suriya K., Philip Robinson J., Gowtham S. and Anjanadevi V.

Antagonistic activity of microbes has been the reason for the failure of many in-dwelling medical devices. These microbes form an integrated behavior of biofilm formation with each other which needs to be overcome for successful implantation. In the present investigation, C. aromatica, a medicinal herb is evaluated for its anti-biofilm activity against gram positive and gram negative bacterial strains using the standard procedure of diffusion method, minimal inhibitory concentration and micro-titer plate assay. The results of this study reveal that gram-positive strain is effectively inhibited by the extract than the gram-negative strain through the MIC at the range of 50-100µg while the bacterial kinetic assay further supported the results of MIC and MBC. The current study has supported the idea of using natural products as an efficient drug for indwelling medical device coronary stent.

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The analysis of sequence variations and expression pattern of Bra-or, controlling the orange head character in Chinese cabbage

Zou Chunlei, Zhang Xi, Liu Aiqun and Feng hui

The orange head Chinese cabbage is widely welcomed by consumers because of its special color. This special character is due to enrichment in carotenoids and is controlled by a single recessive gene, Bra-or, encoding a carotenoid isomerase. In this study, we chose a white variety and six orange varieties of Chinese cabbage to analyze the sequence variations and found that 88-bp and 7-bp deletion occurred in the promoter region and 6-bp deletion happened at the first exon in all six orange varieties. Surprisingly, the frame-shift mutations at the thirteen exon which made the premature termination of translation only appeared in three orange varieties: 07A163, Ju65 and Ju62. qRT-PCR was used to analyze the expression of related genes on carotenoid biosynthesis in Chinese cabbage. The expression levels of Bra-or gradually reduced from outer to inner leaves in the heading stage and the lowest level appeared in the inner leaves of 07A163. The related genes on carotenoid biosynthetic showed significant differences in the heading stage. The results of this study have an important role in understanding the function of Bra-or in controlling the orange head character and provide evidence for further research.

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Isolation, Purification and Characterization of Low Molecular Weight Trypsin from Freshwater prawn Macrobrachium malcolmsonii

Annamalai Asaikkutti, Periyakali Saravana Bhavan and Madhayan Karthik

Trypsin from hepatopancreas of Monsoon river prawn Macrobrachium malcolmsonii was purified to homogeneity using ammonium sulfate precipitation and a series of chromatographies including diethylaminoethylsepharose and soybean trypsin inhibitor sepharose 4B columns. Trypsin was purified to 37.9-fold with a yield of 14.2%. Based on native-polyacrylamide gel electrophoresis (PAGE), the purified trypsin proves a single band. The molecular weight of the trypsin was estimated to be approximately 19kDaby using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal pH and temperature of the purified enzyme for α-N-benzoyl-dl-arginine-p-nitroanilide (BAPNA) hydrolysis were determined to be 8.0 and 50˚C respectively. Trypsin enzyme was stable over a broad pH range from 7.0–11.0 at heat treatment up to 50˚C. The activity was strongly inhibited by soybean N-ρ-tosyl-L-lysine chloromethyl ketone. Purified trypsin had Michaelis–Menten constant (Km) and catalytic constant (kcat) of 0.48mM and 21.15s−1 respectively when BAPNA was used as the substrate. Trypsin with high kcat indicated its high capacity of hydrolysis and it could serve as a promising protease.

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Cluster analysis of SELEX-based candidate aptamers for human C-reactive protein

Yu Xinliang

For the SELEX experiment, a practically important question is how to select candidate aptamers from SELEX products for further affinity determination. In this paper, hierarchical cluster analysis was adopted to create clusters for candidate DNA aptamers that bind CRP (human C-reactive protein) target. During cluster analysis, two molecular descriptors were used as characteristic variables, the median clustering method was used to calculate the distance between two clusters and the squared Euclidean distance is adopted to measure the degree of similarity between two cases (or clusters). 4609 candidate aptamer sequences for CRP target were divided into two clusters. The cluster results for nine DNA sequences whose binding affinities have been characterized and were tested to be in accord with the experimental studies. The feasibility of applying hierarchical cluster analysis to select candidate aptamers from SELEX products for further affinity determination has been demonstrated.

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Principal component and cluster analysis of indigenous tomato genotypes based on morphological indicators

Meena Om Prakash and Bahadur Vijay

The exploration of genetic diversity is a pre-requisite in any breeding programme for effective selection of superior genotypes. Hence, a study was conducted on thirty genotypes of tomato collected from Indian Institute of Vegetable Research, Varanasi and Vegetable Research Station, Junagadh, India to assess the value and magnitude of genetic divergence using Mahalanobis D2 statistics. Wide genetic diversity was observed among the genotypes which were grouped into six clusters by Euclidean method based on D2 values. The clustering pattern indicated that there was no association between geographical distribution of genotypes and genetic divergence. The intra-cluster distance was maximum for cluster V and minimum for cluster III. The maximum distance at inter-cluster level was between cluster III and cluster VI followed by clusters I and VI. Therefore, selection of divergent parents based on these cluster distance would be useful in selecting genotypes for hybridization and formulating a comprehensive strategy to develop superior hybrids or better segregants in tomato. On the basis of mean performance of different clusters, genotypes having maximum flower clusters plant-1, flowers plant-1, radial diameter of fruit and early flowering were observed in cluster III. Genotypes having maximum fruit yield plant-1 along with maximum fruits plant-1, fruit set percentage, fruit weight, polar diameter of fruit, TSS, ascorbic acid and minimum leaf curl incidence percentage were recorded in cluster I. The genotypes of the cluster I for highest mean yield plant-1 along with minimum leaf curl incidence percent can be utilized as donor parent for enhancing the yield and resistance to leaf curl virus of other genotypes grouped in a cluster in F1s and can be fixed by selecting transgressive segregants followed by continued selection in advance generations which may lead to development of high yielding varieties with desired component characters. Principal component (PC) analysis depicted first four PCs with Eigen-value more than one contributing 72.924% of total variability for different traits.

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Anti-obesity Activities of Aronia melanocarpa Nero from Low Temperature Soaking Extraction Process

Lee Hyeon Yong

It was proven that Aronia melanocarpa Nero extract from a simple soaking extraction with 70% ethanol at room temperature had excellent anti-obesity effects. There were only 12.5 g of weight gained by the high fat diet mice after 6 weeks of feeding with 100 mg kg-1 of the extract while approximately 17.5 g of weight were gained by the mice on the high fat diet only and 8.2 g of weight were gained by the control group fed with only the normal diet. However, interestingly, there was minimal weight loss observed by feeding 200 mg kg-1 of the extract, compared to 14 g of weight gained by feeding 50 mg kg-1 of the extract. The group that received 100 mg kg-1 of the extract also showed the least weight gain in the liver, visceral and epididymal fat, compared to the two other groups treated with 50 mg kg-1 and 200 mg kg-1 of the extract. Similar patterns were observed in the reduction of total and LDL-cholesterol, triglycerides and leptin in the serum of the high fat diet mice while the ratio of HDL to total cholesterol was increased. Therefore, these results prove that the extract from a simple extraction process has the effect of reducing both body weight and serum levels of total cholesterol and LDL-cholesterol when feeding the optimal amount of 100 mg kg-1 of the extract to the mice.

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Fabrication of antibacterial bioplastic sheet using orange peel medium and its antagonistic effect against common clinical pathogens

Mridul Umesh and Kathirvel Preethi

The present research work was initiated in a view for fabrication of antibacterial bioplastic sheets using orange peel medium and to assess its antagonistic effect against common clinical pathogens. A sequential and systematic study was carried out for enhancing PHA production by Bacillus subtilis NCDC0671 through one factor at a time approach. Maximum PHA production was achieved at 37 ˚C (5.311 g/L), pH 7 (1.72 g/L) and incubation period of 72 hrs (1.57 g/L). Screening for best carbon and nitrogen source revealed that maximum PHA was produced using sucrose (3.26g/L) and beef extract (2.1 g/L) as carbon and nitrogen source respectively. Characterization of the extracted polymer was done through FTIR, 1H NMR and DSC analysis. Antibacterial biopolymer sheet was prepared by incorporation of need seed extracts in to PHA sheet through solvent casting method. The developed sheets exhibited promising antagonistic activity against common gram positive and gram negative bacteria. The highest zone of growth inhibition was reported against Staphylococcus aureus (23 mm). The results of the present study suggested that biopolymer sheets incorporated with antimicrobial metabolites could be tuned as active packaging or wound healing material.

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Mycotoxins and associated mycoflora in food and feed stuffs in Jeddah markets: analytical and molecular studies

Moussa Tarek A.A.

Aflatoxins were detected in sixty-four samples out of 171 samples and ochratoxin A (OTA) in thirty five out of 96 samples while zearalenone was not detected in any sample. The highest aflatoxins amount was detected in groundnuts (20.4 µg/kg) but the lowest was detected in walnuts (0.7 µg/kg). The four aflatoxins were detected where the highest B1 and B2 amounts were detected in groundnut, G1 in nuts and for G2 in barley grains. The highest OTA amount was detected in poultry feed while the lowest was detected in barley grains. The fungal isolates were identified morphologically and ITS analysis as Aspergillus carbonarius, A. flavus, A. ochraceus, A. parasiticus and Penicillium verrucosum. The aflatoxins were detected in forty-eight samples out of fifty-three A. flavus, this means there were five isolates which are non-alfatoxigenic and on the contrary, A. parasiticus was isolated from all sixty-four samples contaminated with aflatoxins. A. ochraceus was isolated from the thirty-five samples contaminated with OTA while A. carbonarius and P. verrucosum were isolated from twenty and ten samples out of the thirty-five contaminated with OTA respectively. The three studied genes were expressed in A. parasiticus and A. flavus; AflD was the highest expressed gene in A. parasiticus while the lowest was AflR in A. flavus. The two OTA genes studied were expressed in the three fungal isolates. The expression of otapks gene was almost the same level in A. carbonarius and A. ochraceus while the highest expression level of otanps was in A. ochraceus.

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Genotypic study of eighteen Chickpea (Cicer arietinum L.) against Fusarium oxysporum f.sp. ciceri with the help of STMS marker using Capillary electrophoresis and Seed storage protein profiling

Patel C. V., Ramani H.R. and Gajera H.P.

Genetic variability was determined with the help of molecular marker (Sequence tagged microsatellite site) and biochemical marker (SDS-PAGE) in chickpea (desi and kabuli) genotypes against Fusarium wilt. Out of 180 STMS markers, 39 gave significant result and from these TR-60, TR-24 and TA-53 generated unique specific allele of 298, 365, 380 bp respectively as a result of capillary electrophoresis to discriminate desi resistant WR 315 genotype. STMS markers are great help to cluster the genotypes based on desi and kabuli and distinguished resistant genotype in each group. On the other hand, SDS protein profiling of seeds is evident about ~66 KDa protein alleles for discriminating of resistant desi WR 315, JCP 27 and kabuli KAK 2.

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