Research Journal of Biotechnology

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Research Journal of Biotechnology





Phytochemical diversity of Ziziphora clinopodioides Lam. in XinJiang, China

Xu Haiyan, Ding Wenhuan and Tian Shuge

This investigation aimed to phytochemically characterize Ziziphora clinopodioides Lam. from 18 populations in XinJiang, China. Volatile components such as essential oils were analyzed using gas chromatography (GC) and GC-mass spectrometry, the contents of the total phenolic (TPC), flavonoid (TFC), triterpenoid (TTC), free amino acid (TFAAC) and polysaccharide (TPSC) were measured by visible spectrophotometry and the levels of caffeic acid, rosmarinic acid, diosmin, linarin, and pulegone were detected by high-performance liquid chromatography (HPLC). Next, the principal component analysis (PCA) and cluster analysis (CA) methods were used to analyze the genetic diversity of the Z. clinopodioides Lam. germplasm resources from XinJiang province according to the major chemical composition. According to the research, 6 principle components were extracted from the essential oils using PCA and the cumulative contribution rate reached 85.196%. According to CA, both the oils and 10 specific compositions of 18 samples could be classified into 3 clusters and the classification results were broadly consistent with the regions. The results of this research will provide a reliable basis for resource distributions and quality evaluations of Z. clinopodioides Lam.

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Antioxidant Properties derived from Stromata of Xylaria species collected from Tropical Forest in Thailand

Orachaipunlap Krittapong, Suwannasai Nuttika, Chutipaijit Sutee, Whalley Anthony J. S. and Sihanonth Prakitsin

Fungi in the genus Xylaria are common wood decay fungi. They have been shown to be potential sources of novel natural products for utilization in medicine, agriculture and industry. In this study, stromata of Xylaria spp. were collected from Trat Agroforestry Research and Training Station. Fresh specimens of Xylaria spp. were cultured on Potato Dextrose Agar (PDA) for 14 days and then transferred to Malt Extract Broth (MEB). All isolates were cultured under static condition for 6 weeks and extracted with ethyl acetate. Antioxidant properties of stromatic Xylaria spp. were determined in vitro by ABTS and DPPH assays. Free radical scavenging was recorded by Trolox equivalent antioxidant capacity (TEAC) in ABTS assay and EC50 in DPPH assay. The ethyl acetate extracts of four stromatic Xylaria spp. (TR25, TR34, TR46 and TR51) showed strong activities against both ABTS (TEAC 20.70.08, 15.160.03, 110.03 and 19.660.11 M Trolox respectively) and DPPH assay (EC50 36.920.96, 70.531.04, 1022.67 and 49.081.74 L respectively). The correlation coefficients from regression analysis showed a positive correlation between total phenolic/flavonoid contents and TEAC for ABTS assay of all crudes extracted and showed a negative correlation between total phenolic/flavonoid contents and the EC50 for DPPH radical scavenging. Moreover, GC/MS results are indicated the various phenolic compounds in selected crude extracts. In conclusion, cultures obtained from stromata of Xylaria spp. may have potential as natural antioxidant products.

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Treatment of TCE-contaminated Groundwater using In Situ Potassium Permanganate Oxidation: Effects and Kinetics Evaluation

Liang S. H., Wang S. Y., Chang Y.M. and Kao C. M.

The objectives of this bench-scale study were (1) to determine the optimal operational parameters and kinetics when potassium permanganate (KMnO4) was applied in situ to oxidize and remediate trichloroethylene (TCE)-contaminated groundwater and (2) to evaluate the effects of manganese dioxide (MnO2) on the efficiency of TCE oxidation. Approximately 90% TCE removal was observed within the first 275 to 6,880 min for molar ratios of TCE to KMnO4 ranging from 2.53 to 19.63. The calculated second-order reaction rate matched the observed data well. The addition of low doses of KMnO4 could minimize the amount of MnO2 production for a given reaction time. Further detailed investigations of the effect of molar ratios of KMnO4 to TCE were on the removal efficiencies and completeness of the TCE oxidation was warranted for successful application of the controlled-release KMnO4 technique in practice.

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Synthesis and Chracterization of Folic Acid conjugated ZnO/ Silica Core-shell as Cancer Therapeutic and Imaging Agent

Molahasani Nassibeh, Farhadyar Nazanin, Seyed Sadjadi Mirabdullah, Shabadra R. and Gargh S.L.

As a new strategy of combined application of ZnO nanorods with anticancer doxorubicin drug (DOX), we synthesized folic acid conjugated ZnO nanorods (NRs) for targeted drug delivery application purpose. ZnO NRs was synthesized by hydrothermal method and coated with silica layer using tetraethoxysilane (TEOS) and aminopropyltrimethoxysilane (APTMS) at room temperature. Such a surface modified and functionalized samples was then leached in folic acid (FA) as a targeting agent to leave a folate targeted nanostructure. Characterization of the samples was carried out using X-ray powder diffraction (XRD), Fourier Transform Infrared spectroscopy (FTIR) and UV/Visible technique. Field Emission Scanning Electron Microscopy and Energy Dispersive X-ray (FESEM-EDAX) analysis were used to study morphology, distribution and composition of the prepared samples. The results revealed maintenance and chemical bonding of the residual functional groups of folic acid on the nanocarrier’s surface and allowed assembled nanocarrier to be used as a site specific delivery of chemotherapeutic agents.

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Genetic diversity in Xuan nha pine (Pinus armandii subsp. xuannhaensis L.K. Phan)

Nguyen Minh Tam, Phan Ke Loc and Vu Dinh Duy

Pinus armandii subsp. xuannhaensis L.K. Phan is a new five needle pine discovered recently from Xuan Nha Nature Reserve. The subspecies is considered as a narrow endemic to Vietnam and is assessed as endangered. The genetic variation of Pinus armandii subsp. xuannhaensis in Vietnam was investigated on the basis of fifteen ISSR primers. One hundred and seventeen sampled trees from five populations representing the species in Xuan Nha Nature Reserve were used as material for the study. The ISSR data indicated low genetic variability for all the populations, except for Tan Xuan (H = 0.182), with an average of 0.114 and high value at the species level, an average of 0.302. Genetic differentiation among populations was high (Gst = 0.6471) indicating limited gene flow (Nm = 0.273).

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Assessment of genetic diversity using SSR and candidate genes markers among rice accessions (Orysa sativa L.) in relation to grain micronutrient (Iron and Zinc) content

Suma M. R. and Shashidhar H. E

Assessment of genetic diversity in a crop germplasm is a vital part of plant breeding. DNA markers such as microsatellite or simple sequence repeat markers (SSR) and candidate genes (CG) have been widely used to estimate the genetic diversity in rice. Out of 50 rice SSR primer and CGs pairs studied, 47 showed polymorphism on screening in 12 rice genotypes. The trend of diversity was estimated based on the number of alleles (Na), Nei’s genetic diversity index (He) and polymorphism information content (PIC). A total of 141 alleles were identified averaging 2.82 alleles per locus. PIC varied from 0.24 (RM5707) to 0.71 (RM1089 and RM264). The Nei’s genetic diversity (he) varied from 0.28 (RM5707) to 0. 68 (RM144), the average number of he value estimated in present study is 0.53. The NTSYS-pc UPGMA tree cluster analysis led to the distribution of twelve rice varieties into two major groups, I and II. Azucena and IR64 had minimum similarity coefficient of 0.122. Similarity coefficient values indicated that Azucena and IR64 are highly diverse which may be used in further breeding programme for crop improvement.

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Enhancement of Skin Anti-inflammation Activities of Codonopsis lanceolata through Stepwise Steaming Process associated with High Pressure Extraction

Choi Woon Yong and Lee Hyeon Yong

The aim of this study was to enhance the anti-inflammatory properties of C. lanceolata through a stepwise steaming process including a high pressure extraction at 300 MPa for 30 min. First, the extract from the stepwise streaming of C. lanceolata by high pressure extraction (SHC) showed the highest reducing power at 3.05 (O.D) compared with 2.01 (O.D) in the convention-al water extraction at 100oC. The SHC extract most strongly inhibited NO production from RAW 264.7 cells, requiring a final concentration of only 6.33 μM whereas 9.64, 6.62 and 10.74 μM were required from only steamed C. lanceolata (SC), high pressure extracted C. lanceolata (HC) and fresh C. lanceolata (FC) respectively. The SHC extract also significantly inhibited LPS-induced PGE2 production from macrophages at concentrations as low as 15%. Hyaluronidase activity, a key indicator of skin inflammation, was reduced to 50% in both the SHC and FC. It was proven that the high anti-inflammatory activities found in the SHC can be attributed to the anti-oxidative properties of phenolic acids. For example, 27.03 μg/mg of gallic acid and 12.86 μg/mg of vanillic acids were extracted in the SHC process. Notably, these bioactive substances prevent the generation of active oxygen in the skin as well as the oxidation of the skin. It was also found that the SHC can effectively suppress iNOS and COX2 expression levels which are strongly related to skin inflammation. Suppressing the expression of these genes will eventually suppress inflammation and improve skin immunity through anti-oxidative processes.

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Molecular characterization of plant growth promoting bacterial strains from Pinus roxburghii L. rhizosphere

Younis Tayyaba and Faisal Muhammad

Present study deals with the isolation and characterization of twenty five bacterial strains from the rhizosphere of Pinus roxburghii growing in three different geographical regions (Lahore, Islamabad and Mansehra) of Pakistan. Out of twenty five isolates, eight (32%) solubilized tri-calcium phosphate (TCP), nine (36%) produced plant growth hormone indole acetic acid (IAA), five (20%) produced anti-fungal metabolites and ten (40%) produced siderophores. On the basis of 16S rRNA gene sequences, thirteen Gram-positive (52%) and twelve Gram-negative (48%) isolates were assigned to ten genera and twenty one species. Of these genera, Bacillus (five species) made up the largest complement followed by Pseudomonas (two species). The Gram-negative rhizobacteria were distributed in two phyla: Proteobacteria (α-proteobacteria, γ-proteobacteria) and Flavobacteria. Plant growth experiment study revealed that strains (Comamonas sp., Enterobacter sp., Bacillus simplex, Bacillus sp., Pantoea sp. and Pseudomonas aeruginosa) were able to enhance wheat root length to a significant level (up to 47%) as compared to non-inoculated control.

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Protoplast isolation and fusion in cultivated Cajanus cajan (L.) Millsp. and wild species of Pigeonpea

Parihar A., Pachchigar K. and Zaman Mariya S.

Pigeonpea (Cajanus cajan (L.) Millsp.) is an important grain legume crop of rainfed agriculture in the semi-arid tropics and second most important pulse crop grown in India. Present work has been attempted to isolate, characterize, fuse and culture leaf derived protoplasts of different 10 lines of pigeonpea comprising of 8 wild species and two predominant varieties of cultivated species of pigeonpea (Cajanus cajan (L.) Millsp.) i.e. GT-101 and GTR-11. The isolated protoplasts of all the eight diverse wild species of pigeonpea were attempted to be fused with two cultivated varieties of pigeonpea- GT-101 and GTR-11. Protoplasts were successfully isolated from cultivated and wild pigeonpea. Several combinations and concentrations of hydrolytic enzymes with PEG were used for protoplast isolation and fusion, respectively. The incubation in an enzyme mixture containing 1.0 % cellulase, 0.15% macerozyme and 2.0% pectinase for 18 hr at 25ºC in dark produced good yield and viability. However, the best response was obtained with species Rhyconsia minima. Among different combinations of PEG concentrations (28, 35, 40%) tested in general, 28% PEG treatment produced the highest fusion frequency. Purified protoplasts cultured in agarose solidified MS medium were tested. A total of four hormones 2,4 –D, kinetin, BAP and NAA were used with different concentrations. It has been observed that proto colony was achieved within 5-6 days of culture and within 9 to 10 days, clusters of 20 to 30 cells were obtained. The maximum calli formation was observed in 2,4-D whereas it was minimum under NAA. Further, for confirmation of regeneration in proto calli, this was observed under microscope which clearly showed regenerated cell wall and vacuolated and clustered shape of parenchymatous cell which could confirm growing calli from proto calli.

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Transcriptome profile and differentially expressed genes analysis in winter wheat under cold stress conditions

Dongwei Xie and Zhuofu Li

Winter wheat can regrow surviving the cold winter in extremely cold areas and this type of cold resistance is a very attractive quality. Very little is known regarding the molecular mechanism of cold resistance in winter wheat, particularly the identity of the cold resistance genes. In this study, RNA was extracted from the crown of winter wheat varieties with different cold resistances subjected to various low-temperature treatments. Using the Solexa/Illumina sequencing platform, 60 million sequencing reads were obtained and these reads were assembled into 80,704 unigenes. Based on the method of known protein similarity search, we acquired 51,929 sequences that were consistent with the standard E-value cut-off of 10-5. Additionally, 22,724 sequences were annotated by gene ontology (GO) term, 31,964 sequences were annotated by Swiss-Prot, 18,764 sequences were clustered into 43 types by Clusters of Orthologous Groups classifications (COG) and 29,553 sequences are assigned to 125 pathways by the Kyoto Encyclopedia of Genes and Genomes pathways (KEGG). Furthermore, transcription factor genes involved in cold and dehydration resistance are more highly expressed in Dongnongdongmai 1, which is cold resistant, compared to Jimai 22, which is cold sensitive. The expression of genes in Phenylalanine metabolism, Alpha-linolenic acid metabolism, Gluathione metabolism, as well as Starch and sucrose metabolism pathway was triggered by LT. In addition, these pathways had difference between the two winter wheat varieties. We performed cluster analysis for the differentially expressed genes. Eight genes were randomly selected for expression quantity validation by quantitative RT-PCR. These results of the gene expression pattern analysis under three low temperature treatments are essentially same as the DGE data. Summarily, we obtained an extensive transcriptome dataset from winter wheat, a non-model whole genome which identifies winter wheat cold resistance genes under low temperature conditions.

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Molecular cloning, sequence characterization and expression pattern of BURP domain-containing protein 17 gene from watermelon (Citrullus lanatus)

Peng Lei

The complete mRNA sequence of the watermelon BURP domain-containing protein 17 gene was amplified through rapid amplification of cDNA ends (RACE) method. The full-length mRNA was 1,085 bp containing a 996 bp open reading frame which encodes a protein of 331 amino acids. Sequence analysis revealed that watermelon BURP domain-containing protein 17 protein shares high homology with the BURP domain-containing protein 17 of cucumber (82%), muskmelon (82%), apple (58%), soybean(57%)and wine grape (54%). Phylogenetic analysis revealed that watermelon BURP domain-containing protein 17 gene has a closer genetic relationship with the BURP domain-containing protein 17 gene of cucumber and muskmelon. Tissue expression profile analysis was carried out and results indicated that the watermelon BURP domain-containing protein 17 gene was highly expressed in root and stem, moderately expressed in flower and leaf, and weakly expressed in fruit. These results established the primary foundation to understand the watermelon BURP domain-containing protein 17 gene.

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Economic Feasible Evaluation of Biogas Production from Napier Grass

Sawasdee Vanatpornratt and Pisutpaisal Nipon

Napier grass, a fast growing tropical plant, can grow up in drought and dry conditions. Due to its abundance in nutritional compositions, the Napier grass is the ideal, non-food crop feedstock for biogas production. The anaerobic digestion of Napier grass with varying the grass content in the range of 5 - 15 % (w/v) was carried out in 500 mL batch reactors. The fresh grass was harvested after 45-d plantation, grounded in 0.5 to 1 mm diameter size before fermentation in mesophilic condition. Solid content of the grass influences the methane content, yield and production rate. The optimum condition for the biogas production was found at 5% solid concentration. Methane content, yield and production rate were 53%, 0.122 L CH4/g TVS, 4.8 mL/h at the optimum condition. Economic analysis of the biogas obtained from the fermentation as a substitute of liquid petroleum gas (LPG) showed benefit/cost ratio (B/C ratio) greater than one suggested that the Napier grass is an economic feasible feedstock for biogas production.

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Identification of temperature stress-induced differentially expressed genes of tall fescue leaves

Lee Sang-Hoon, ChoiGi Jun, KimKi-Yong, JiHee Chung, Park Hyung Soo, Lee Dong-Gi and Lee Ki-Won

In the present study, we investigated temperature stress-responsible differentially expressed genes (DEGs) using annealing control primer (ACP)-based differentially display reverse transcription-polymerase chain reaction technique in tall fescue leaves. Using 120 ACPs set, 10 genes were up or down regulated under cold or heat stresses followed by sequencing to investigate functional information. In cold stress, each three DEGs were up and down regulated respectively. In the meantime 6 DEGs were up regulated and 2 DEGs were down regulated under heat stress. Of these DEGs, pathogen related protein10-1, iron-sulfur assembly protein lacA, ABC transporter C family member 14, lipoxygenase 11, protein cornichon-like protein and uncharacterized protein LOC100823022 were identified. These up-regulated genes may play a pivotal role in effective resistance mechanism under temperature stress in tall fescue.

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The Difference Expression Profiles of MicroRNA-206 during the Growth Process of Goat

Ling I. Y. H., Ding J. P., Wang L. J., Liu Y., Zhang Y. H., Zhang Z. J. and Zhang X. R.

Many miRNAs have been reported for their important roles in developmental processes in various animals but there is limited information about goat miRNAs. In this study, the temporal and spatial expression patterns of miR-206 in skeletal muscle (longissimus dorsi) from Anhui white goat and Boer goat were assessed using Real time quantitative PCR (qPCR) method. In addition, the association between miR-206 expression and the muscle development was analyzed. The expression of miR-206 has tissue and time specificity. The expression level in longissimus dorsi muscle from 360 days goats was the highest while the expression level in neonate was the lowest. In 180 days goats, miR-206 was abundantly expressed in muscle and heart, moderately in liver and kidney and weakly in other collected tissues. The results indicated that miR-206 may be involved in myofibre proliferation and differentiation and further contribute to skeletal muscle development and phenotype with different genotypes. The present results provided the theory base for study of miR-206 function.

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