Research Journal of Biotechnology

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Vol. 8(11) November 2013

Milk protein genetic variants in Romanian Spotted, Holstein Friesian and Montbéliarde cows and some correlations with milk parameters

Grădinaru A.C.*, Ilie Daniela Elena and Creangă Şt.

Milk samples of 184 Romanian Spotted, 189 Holstein Friesian and 62 Montbéliarde cattle from Romania were phenotyped for caseins and whey proteins variants, using IEF and PCR-SSCP techniques. The allele and genotype frequencies at each locus were calculated using PopGene version 1.31. Additionally, each locus within the three populations was tested for Hardy – Weinberg Equilibrium. The alpha-s2-casein and alpha-lactalbumin proteins showed monomorphism in all three breeds, with A and B allele, respectively. The other proteins showed polymorphism with B and C alleles of alpha-s1-casein; A1, A2, B, C and I alleles of beta-casein; A, B and E alleles of kappa-casein; and A, B and D alleles of beta-lactoglobulin. Hardy-Weinberg equilibrium was observed for all investigated protein variants within the three breeds. The haplotype frequencies including alpha-s1-, beta- and kappa-caseins were calculated with the EH Programm package. The B-A2-A haplotype was the most prevalent in the Romanian Spotted and Holstein Friesian populations, whereas the B-A2-B haplotype was the most prevalent in the Montbéliarde cows. The effects of milk protein genotypes on the milk production traits as well as protein and fat percentages in Romanian Spotted cattle were evaluated using SAS procedure. A significant influence of genotypes on milk parameters was found only for beta-casein for milk (p<0.01), protein and fat yields and protein percentages (p<0.05). The A1A1 genotype was associated with more milk yields and higher protein and fat contents than other genotypes. The A2A2 and the group of A2B and A2C genotypes were associated with the highest protein rates.

Research on the Antioxidant Activity of Phenols and Active Oxygen Metabolism during Development of Brazil Banana

Hui-Gang Hu

Banana (Musa spp.) is economically important fruit throughout the world. In the present study, the antioxidant activity of phenols and active oxygen metabolism during the development of banana fruit were investigated. Total phenolic content and total antioxidant activity (T-AOC) were significantly correlated in both peel (R2 = 0.849) and pulp (R2 = 0.884); the peroxidase (POD) activity was significantly correlated with total phenolic content in both peel and pulp, suggesting the POD is an important enzyme for active oxygen metabolism in banana fruit. Also a strong correlation (R2 = 0.891) between T-AOC and total flavonoid was observed in peel. The total phenolic was more abundant in peel than in pulp and the T-AOC in peel was significantly higher than that in pulp during the fruit development. Furthermore, the T-AOC in the early developmental stage was stronger than that in the later developmental stage in peel. These results suggested that banana peel and immature fruit which are rich in phenolics might provide good source of antioxidants.

Effect of Probiotics along with selected Prebiotics against Respiratory Tract infecting Pathogens

Usha G.*, Ravi D. and Parthasarathy R.

Probiotics are harmless bacteria that provide equilibration of the intestinal flora and hence have a positive effect on the health of the consumer. Probiotics are live microbes that can be formulated into many different types of products including foods, beverages, drugs and dietary supplements. Probiotics had high antagonistic activity, antimicrobial activity and enzymatic activity. Prebiotics had the ability to support the growth of probiotics. The probiotic strain Lactobacillus plantarum and Lactobacillus rhamnosus were isolated and tested for antagonistic activity. Among the ten prebiotics used, Aloe vera, Cichorium intybus and Borassus flabellifer showed higher antagonistic effect against Staphylococcus aureus and Streptococcus pneumoniae and in addition, suitable prebiotics for probiotic efficient growth was found. Considering the antimicrobial activity, it was found that Staphylococcus aureus growth was fully suppressed in Aloe vera, Punica granatum, Cichorium intybus. Streptococcus pneumoniae growth was fully suppressed in Aloe vera, Jersaleum artichoke and Cichorium intybus. In all of the three tests, antagonistic, antimicrobial and enzymatic effect were studied and the result revealed that highly significant control against Staphylococcus aureus and Streptococcus pneumoniae was given by Lactobacillus rhamnosus. Probiotics reduce the risk of respiratory disease probably due to their role in suppressing the activity of certain bacterial enzymes with the production of bacteriocins. Thus probiotic treatment will offer a promising alternative to the use of antibiotics in healthcare.

Micropropagation and chloroplast isolation from in vitro of Aloe Vera plants

Hoang T. K. H.

The plant, Aloe vera L., belongs to the Liliaceae family and is well-known as an important pharmaceutical plant that is used for investigating and creating medicinal and cosmetic compounds. Notably, this specie displays all of the physiological features of CAM (Crassualacean acid metabolism) photosynthesis along with exhibiting net nocturnal CO2 uptake. Among CAM plants, A. vera is one of numerous phosphoenolpyruvate carboxykinase (PCK)-CAM plants which contain significant activities of PCK with lower levels of malic enzyme (ME). Generally, A. vera requires the operation of malate dehydrogenase (MDH) to convert malate to oxaloacetate (OAA). OAA is further converted to phosphoenolpyruvate (PEP) and CO2 by cytosol PCK. Although the micropropagation of A. vera process has been well-studied, very little attention has been given to the role of the A. vera chloroplasts from in vitro plants during the CAM photosynthesis. In doing this research, we produced the in vitro plants of A. vera by creating a tissue culture and then we used the leaves of in vitro plants to isolate pure, intact chloroplasts by the use of Percoll density gradient centrifugation.

Role of Fungi and Fungal Extract on Strychnos Potatorum Seed Germination

Srikanth Kagithoju, Althaf Hussain Sk, Vikram Godishala and Rama Swamy Nanna*

Strychnos potatorum seeds have slow and erratic germination, thus seed priming methods like treatment with fungi and fungal extract were applied to enhance the germination efficiency. In the present investigation, two dominant seed mycofloral members - Aspergillus niger, Pencillium citrinum were isolated and identified from fallen fruits using blotter paper method. The role of these fungal members and their extracts individually and in combinations in germination was investigated. Germination efficiencies were increased with use of fungi and fungal extracts. More percentage (65%) of germination was found when P. citrinum culture extract was used than in control seeds (45%) followed by in both fungal extracts combination (62%). Infestation with these fungal members individually or in combination or treatment with extracts resulted in the decrease in dormancy period and increased the germination. The present findings clearly showed that seeds are facultative dependent on fungal members as they showed germination even in the absence of fungi but with less efficiency. These two fungal cultures and fungal extracts hence can be used for breaking seed dormancy and enhancing germination percentage in S. potatorum which is endangered and medicinally important. Thus, this pre treatment method can be adopted for in situ conservation of S. potatorum.

Molecular Cloning, Sequence Characterization and Gene Expression Profile of a Novel Water Buffalo (Bubalus bubalis) Gene: Annexin A9 Gene (ANXA9)

Song Shen, Huo Jinlong and Miao Yongwang*

The complete coding sequence (CDS) of water buffalo Annexin A9 (ANXA9) was amplified and identified using the reverse transcription-polymerase chain reaction (RT-PCR) based on the conserved sequence information of cattle or the expressed sequence tags (ESTs) of other Bovidae species. Sequence analysis revealed that the CDS of water buffalo ANXA9 encodes an enzyme of 345 amino acid residues with a deduced molecular weight of 38.45 kDa and a PI of 8.74. The deduced amino acid sequence of water buffalo ANXA9 shares 98, 83, 86.4, 76.5, 77, 82, 83.8 and 45.7% identity with its homologous sequences of cattle, horse, pig, mouse, rat, chimpanzee, human, African clawed frog respectively. The phylogenetic tree analysis based on the CDS of ANXA9 gene showed that water buffalo has a closer genetic relationship with cattle than with other species. The ANXA9 was widely expressed in the buffalo tissues examined, being high in the pituitary gland, muscle and brain; moderate in the heart, spleen, liver, mammary gland and lung; weakly expressed in the small intestines and skin and not expressed in the adipose tissue and stomach. The results of the present study will establish a foundation for further insights into this novel water buffalo gene.

SOD, CAT, GR and APX Enzyme Activities in Callus Tissues of Susceptible and Tolerant Eggplant Varieties under Salt Stress

Yaşar Fikret, Talhouni Manar, Ellialtioğlu Şebnem*, Kuşvuran Şebnem and Uzal Özlem

When plants are subjected to environmental stresses such as temperature extremes, drought, salinity or heavy metal toxicity, they produce reactive oxygen species ROS causing oxidative damage. Plants with high levels of antioxidants, either constitutive or induced, have been reported to have greater resistance to the oxidative damage. In our previous studies, we examined the relationship between antioxidant enzymes and salt tolerance in the leaves of the eggplant seedlings of two salt-tolerant genotypes (Burdur Bucak-BB and Mardin Kızıltepe-MK) and two salt-sensitive genotypes (Giresun-GI and Artvin Hopa-AH) and we found that the salt-tolerant varieties contained significantly higher constitutive levels of catalase and induced levels of SOD, APX and GR. SOD, CAT, GR and APX enzyme activities were determined in callus tissues. Under salt stress at the 8th day, significant differences were found in the aspect of SOD activity between salt-tolerant and salt-sensitive eggplant varieties. Concerning CAT, GR and APX activities, it is found that the 4th day calculations were significant and gave a high correlation with the tolerance levels of genotypes. The data indicated a strong correlation between antioxidant enzyme activity and the ability of eggplant callus tissue to grow on media amended with NaCl. The salt-induced increase in antioxidant enzyme activities in callus tissue was found at the highest level in salt tolerant eggplant varieties. The genotypical differences in salt tolerance observed at whole plant level experiments were also apparent in callus cultures of eggplant. These results show that callus cultures can be useful for the screening programs to determine the salt tolerance level in eggplant.

Reproduction biotechnologies in Mangalita breed boars

Ciornei Ş.G., Roşca P.*, Drugociu D., Mare M., Nechifor F. and Ibănescu I.

One of the breeds of pigs that led to the development and rearing of pigs in Carpathian area is Mangalita. In order to prevent the extinction of this race, it is still reared in special centers, representing genetic heritage. Being an old breed, it represents useful gene pool on which current and future biotechnology can be applied. This study aimed to address the main reproductive parameters of this breed compared to improved breeds. Thus, in Mangalita group of boars the following semen indices were obtained: average volume of 202.8 mL, concentration of 213x106 spermatozoa/mL, (43.9 x109/ejaculate), total mobility of 82% with a rate of progressive movements of 42%. After artificial insemination (AI), the fecundity was 63.3%, parturition rate was 100% and prolificacy was 8.12 piglets / farrowing. The values of spermogram, fecundity and prolificacy in Mangalita were in limits of the species. Some indicators were lower than in the improved breeds while others were alike or even higher. The study shows that the obtained reproductive indices guarantee the suitability of this breed for intensive exploitation and the opportunity to apply the breeding biotechnology of this rustic breed.

Bioinformatics Analysis of Zea mays NAC Protein

Sun Qingpeng1*, Yongkun Yu, Jun Han1 and Pan Jinbao

A completed cDNA (JF795488.1) was isolated from zea mays and the protein encoded was named as zmNAC which consisted of 282 amino acids and was analyzed by bioinformatics tools. The results showed that zmNAC belongs to the NAC transcription factor and it was a hydrophilic protein which contained 21 potential phosphorylation sites and one N-Glycosylation potential site.

Phytochemistry and biological activities of terpene-rich essential oil (TREo) from Teucrium sandrasicum

Celik Ali*, Ermis Ahmet, Herken Emine N. and Arslan Idris

This study was aimed at evaluating the chemical constituents, antioxidant and antibiotic activity of the TREo (terpene-rich essential oil) of the endemic plant species Teucrium sandrasicum O.Schwarz. The TREo obtained by hydrodistillation using a Clevenger-type apparatus and constituents was analyzed by gas chromatography/mass spectroscopy (GC/MS). The main components of the TREo were detected as β-pinene (41.72%), α-pinene (13.95%), germacrene-D (10.68%) and sabinene (9.99%).Antioxidant capacity of TREo was tested the method Trolox Equivalent Antioxidant Capacity (TEAC) assay in vitro. The antibiotic assay of the TREo on ten pathogenic bacteria including Escherichia coli MC-4100, Citrobacter freundi NRRL-B 2643, Providencia stuartii, Proteus vulgaris NRRL-B-123, Pseudomonas aeruginosa NRRL-B-2679, Enterobacter aerogenes NRRL-B-3567, Bacillus licheniformis NRRL-B- 001, Staphylococcus aureus ATCC-33862 was examined with agar disc diffusion method.

Molecular Characterization of Bacterial Population in the Rumex Patientia Rhizosphere Soil of Jilin, China

Xiaojuan Qi1, Ensi Wang and Xia Chen*

The bacterial diversity in the Rumex patientia rhizosphere soil of Jilin, China was investigated by 16S rDNA-dependent molecular phylogeny. Small subunit rRNA (16S rDNA) from soil metagenome was amplified by polymerase chain reaction (PCR) using primers specific to the domain bacteria and analysed by cloning and sequencing. A total of 83 unique phylotypes were identified and classified as Proteobacterium (43.37%), Bacteroidetes (13.25 %), Acidobacteria (10.84%), Unclassified bacteria (9.64%), Gemmatimonadetes (7.23%), Verruco-microbia (4.82%), Planctomycetes (4.82%), Actinobacteria (3.61%), Firmicutes (1.20%) and Chloroflexi (1.20%).

Effect of process parameters on the performance of novel glutaminase free L-asparaginase from Erwinia aroideae NRRL B-136 under assay conditions

Goswami Rachna, Veeranki Venkata Dasu*, Hegde Krishnamoorthy and Bamnia Meenakshi

Glutaminase-free L-asparaginase is an important therapeutic agent used for the treatment of various lymphoblastic leukemia diseases. In the present study, the performance of L-asparaginase was performed under assay conditions. The deactivation rate constants, half-life of L-asparaginase, thermodynamic parameters viz. ΔH*, ΔS*, ΔG* and activation energy of thermal deactivation of L-asparaginase were calculated. The optimum pH and temperature of the purified L-asparaginase were found to be 8.6 and 57°C respectively. Km and Vmax of purified L-asparaginase were found to be 0.0056 mM and 26.31 IU respectively.

Genetic damage analysis of Autism Spectrum Disorder (ASD) patients of Tamilnadu, South India using Cytokinesis-block micronucleus cytome (CBMN cyt) assay

Rajiv Sundaramoorthy, Kavya M. Rao and Valsala Gopalakrishnan Abilash*

The frequency of micronuclei in peripheral blood lymphocytes of ASD patients were monitored with the CBMN Assay. We have evaluated a random population of 46 children and 5 adults aged 2–24 years with appropriate age and sex matched controls. The patients were first clinically pre-screened with help of physician. Peripheral blood samples were collected from antecubital vein, cultured using RPMI-1640medium and treated with Cytochalasin-B. Harvested cells were stained using giemsa and a total of 1000 BN cells were scored for all the subjects. All the patients were analyzed cytogenetically and categorized clinically using the DSM-IV manual. Patients with PDD, ADHD, MR, AS and both PDD/ADHD, showed significant proportion of DNA damage which was statistically significant when compared with age and sex matched controls. This study shows a significant level of DNA damage in ASD patients when compared to controls. To an extent, environmental, occupational exposures, lifestyle factors, dietary deficiencies and different diseases have also added up to the rise. Important knowledge gap still remains about the characteristics of micronuclei and other nuclear abnormalities, including the basic biology explaining the appearance of various cell types in autism patients which may be addressed by further studies.

Establishment of Regeneration System on Maize Shoot Tip

Zhou Rong, Sun Qingpeng, Yu Yongkun*, Han Jun and Pan Jinbao*

The shoot tips of the maize inbred lines 2193, 7922, 9002 and Zheng 58 were used to culture on MS mediums supplemented with different concentrations of 6-BA and 2,4-D. The regenerated plantlets were obtained after the induction and regeneration of the multiple shoot clumps. After the comparison of different tests, optimal culture conditions for induction of the multiple shoot clumps of different genotypes were made out. The generation of multiple shoot clumps and plant regeneration showed significant differences in varied genotypes of maize. Thus a shoot regeneration system was established initially for the shoot tip meristems of maize.

Morpho-molecular characterization of Indian isolates of Rhizoctonia solani infecting mungbean

Upadhyay B. K., Dubey S.C.,* Singh Ravindra and Tripathi Aradhika

Rhizoctonia solani causes web blight and root rot disease in mungbean. Forty multinucleate R. solani iso1ates from mungbean representing 27 districts from 11 States of India were isolated and morphologically characterized based on their hyphal anastomosis reaction, hyphal width, growth rate, number, size and pattern of sclerotia formation. The isolates were characterized into 7 different anastomosis groups (AGs). The isolates were variable in their growth rate ranging from 0.9 mm to 1.8 mm/h, number of sclerotia per plate (7 to 1900) and the size of sclerotia (0.3 mm to 3.0 mm). Genetic diversity of the isolates was analyzed using random amplified polymorphic DNA (RAPD), inter-simple sequence repeats (ISSR) and simple sequence repeat (SSR) markers. RAPD markers showed 98.5% and ISSR showed 94.1% polymorphism with the band size ranging from 0.1 to 5 kb whereas SSR markers showed 93.6% polymorphism with the bands sizes ranging from 0.1 to 1.5 kb. Combined analysis of all the three markers by unweighted pair group method with an arithmetic average (UPGMA) grouped the isolates into 8 major clusters at 52% genetic similarity and AGs specific sub-clustering was observed. The isolates showed high genetic variability and the molecular groups were partially corresponding to AGs and area of the origin but did not correspond to morphological characters.

Effects of sodium dodecyl sulphate on biooxidation of copper mine tailings by Acidithiobacillus ferrooxidans

Li Dou, Yin Yajie, Li Dongwei*, Song Liyan, Wang Yangqing, Tang Wei and Xu Zhonghui

Biooxidation and bioleaching are disposal methods for copper mine tailings to recover metals and to reduce the environmental threat of toxic elements. However, copper mine tailings contain numerous residual common flotation agents such as sodium dodecyl sulphate (SDS) whose effect on biooxidation remains unknown. In the present work, the effects of SDS on biooxidation copper mine tailings by Acidithiobacillus ferrooxidans were investigated on 5 mg/L and 10 mg/L respectively. The results showed that 10 mg/L SDS completely inhibited A. ferrooxidans growth and caused biooxidation loss due to damage to the cell membrane and changes in mineral surface hydrophilicity. Scanning electron microscope (SEM) observations of the surface of copper mine tailings after 30 days of biooxidation clearly show that aggregates suggest a combination of chemical and biological oxidation during biooxidation of copper mine tailings due to the presence of A. ferrooxidans. The results of the study indicated that A. ferrooxidans could recover from treatment with 5 mg/L SDS but Cu dissolution rate did not improve.