LEAF RUST RESPONSIVE EXPRESSION PROFILING OF GRAS TRANSCRIPTION FACTOR FAMILY IN WHEAT (TRITICUM AESTIVUM L)

Objective: Rusts are among the most important fungal diseases of wheat all over the world responsible for losses in yield ranging from 25% to 90%. Bread wheat (Triticum aestivum L.) is one of the major staple food crops all over the world but is greatly affected by leaf rust. GRAS is a plant-specific stress responsive transcription factor gene family. The objective of the present study is to carry out expression profiling of GRAS TFs during leaf rust pathogenesis.Methods: SOLiD SAGE library preparation. GRAS TFs were mapped to the four libraries using the CLC genomics workbench to study their expression profiles. A Co-expression network of these TFs has been constructed using WGCNA (weighted gene co-expression network analysis).Results: The four libraries have been prepared: S-M, S-PI, R-M R-PI. GRAS TFs were mapped to these libraries, giving different expression profiles of the 63 GRAS TFs. Pearson correlation coefficients were 0.56, 0.34 and 0.24 for R-M vs. R-PI, S-M vs. S-PI and S-PI vs. R-PI respectively. Highest difference in expression of TaGRAS genes was between two libraries S-PI vs. R-PI. TaGRAS genes have been clustered into seven (blue, turquoise, red, green, black, maroon and yellow) different modules in signed correlation.Conclusion: TaGRAS genes which are upregulated during leaf rust might be plays important roles to provide resistance to the plants. The difference in Pearson correlation coefficient indicates that susceptible and resistant-NILs utilize a different set of TaGRAS genes to counter leaf rust pathogenesis. The genes which are clustered together in coexpression network might be expressed together during leaf rust pathogenesis to provide resistance to the plant.Keywords: Leaf rust, Wheat. GRAS, Transcription Factors, SAGE, WGCN

TRANSCRIPTOME-WIDE ANALYSIS OF NFX1 TRANSCRIPTION FACTORS IN WHEAT (TRITICUM AESTIVUM L.) AND THEIR LEAF RUST RESPONSIVE EXPRESSION PROFILING

Objective: The purpose of this study was to identify and characterize NFX1 transcription factors (TFs) in wheat and study their expression profiles in response to leaf rust infection.Methods: NFX1 transcription factors were identified by in silico data-mining, followed by characterisation using different bioinformatics tool. The evolutionary relationship was established by constructing a phylogenetic tree with Arabidopsis NFX1 proteins using Molecular Evolutionary Genetic Analysis (MEGA5). Expression analysis of identified TaNFX1 TFs in wheat was performed using CLC Genomics Workbench.Results: Nine NFX1 transcription factors were identified in wheat. Evolutionary analysis revealed their classification into group 1, 2 and 4 type NFX1 zinc finger. Tag based expression analysis revealed that based on the fold change values, the maximum level of expression was observed in TaNFX1-3 and 7 whereas, the minimum level of expression was observed in TaNFX1-2 in response to leaf rust pathogenesis. Chromosomal localization predicted that identified NFX1 sequences belonged to 3A, 3B, 3D and 7D chromosomes.Conclusion: Using transcriptomic approach nine NFX1 TF proteins are predicted that regulate gene expression in response to leaf rust disease in wheat which has not been reported or studied before. Functional and bioinformatics-based exploration of wheat NFX1 TFs in related monocots might provide subsets of candidate target genes to improve agronomic traits related to biotic stress tolerance.Â

SCREENING AND GROWTH KINETIC STUDIES OF WILD CHLOROPHYCEAN FRESH WATER MICROALGAL SPECIES FOR BIOMASS AND BIOFUEL APPLICATIONS

Objective: Microalgae are studied for decades for various products such as, protein rich animal/fish feed, lipids, pigments, neutra ceuticals, therapeutic agents, primary products and biomass. Lipid content was prime target in most of the research programs for production of biodiesel as an alternate to fossil fuel. Chlorophycean microalgae has the potential to meet all these requirements. The objective of this study was to collect and identify chlorophycean microalgae from various water bodies of Jharkhand State of India and to estimate their total lipid content.Methods: Wild cholorophycean fresh water species from Jharkhand were collected and studied for biomass, total lipid, carotenoids and chlorophyll content. Fourier transform infrared spectroscopy (FTIR) data were obtained for further verification of lipid estimation in all the species. Light microscopy as well as Scanning Electron Microscopy (SEM) was performed to identify the species.Results: The observation revealed two groups of micro algae, among these Scenedesmus sp and Chlorella sp. Showed highest lipid accumulation of 45.1 and 41.5 % respectively, while Legerhemia sp. Showed highest biomass production (21.2 g/l). Productivity/day for an 80K L pond system was calculated by extrapolation of results; that changed the choice of organism to Desmodesmus sp.Conclusion: The microalgae collected from highly polluted sites were efficient enough to yield high lipid (AKS-1/AKS-8) and biomass (AKS-6). The laboratory scale study was extrapolated with mass scale culture data and the choice of organism changed to AKS-16 from AKS-1/AKS-8 (for high lipid content) or AKS-6 (for high biomass).Â

MINING SINGLE NUCLEOTIDE POLYMORPHISM FROM PUBLICLY AVAILABLE ESTS OF BREAD WHEAT (TRITICUM AESTIVUM L.)

Objective: The present study was undertaken to discover Single Nucleotide Polymorphisms (SNPs) in bread wheat with reference to leaf rust disease.Methods: Next Generation Sequencing platform sequencing by Oligonucleotide Ligation and Detection (SOLiD) was performed on four Serial Analysis of Gene Expression (SAGE) libraries of mock and leaf rust pathogen infected near-isogenic lines HD2329±Lr28. CLC Genomics Workbench was used for computational prediction of the SNPs. The predicted SNPs were filtered by Blast using wheat Expressed Sequence Tags (ESTs). The SNP-containing ESTs were annotated, and their expression was checked in response to inoculation of Puccinia triticina.Results: We have identified 191 SNPs from data obtained through the These EST-SNPs participated in various physiological and biochemical processes that influence important traits, such as cell rescue, defense and disease resistance.Conclusion: Very little knowledge exists on SNPs in hexaploid bread wheat (Triticum aestivum L.) because of the difficulty to discern the true polymorphic loci. This study has revealed fast and costs effective approach for SNP discovery which will be helpful in molecular breeding with important agronomic traits.Â

EFFECT OF POLYSORBATE-80 CONCENTRATION ON G-CSF FORMULATION USING LIQUID CHROMATOGRAPHY

Objective: The aim of the study was to find the dual effects of polysorbate-80 concentration on oxidation and aggregation in the formulation of granulocyte colony stimulating factor. Methods: The effect of polysorbate-80 at different concentration was monitored and analyzed using high performance liquid chromatography. Oxidative degradation and aggregate generation was studied using reverse phase and size exclusion chromatography method respectively. Results: With increase in concentration of polysorbate-80 the amount of oxidation as well as aggregate formation increases in a concentration dependent manner. The aggregates present at higher concentration of polysorbate-80 formulation are not found with low concentration or without polysorbate-80. This result shows that higher concentration of Tween-80 force the formation of oligomers and leads to increased level of oxidation of granulocyte colony stimulating factor. Conclusion: The dual effect of aggregation and oxidation of polysorbate-80 on the recombinant granulocyte colony stimulating factor indicate that during formulation development studies it is crucial to evaluate the amount of polysorbate-80 to be used in the formulation

A DE NOVO ASSEMBLY METHOD FOR SHORT SEQUENCE OF SOLID-SAGE READS RESPONSIBLE FOR WHEAT (TRITICUM AESTIVUM L.) LEAF RUST

Objective: Wheat leaf rust is one of the most widespread rust diseases caused by Puccinia triticina Eriks. De novo assembly of short sequence reads in order to understand the molecular phenomenon underlying wheat leaf rust interaction and to assemble differentially expressed genes, resistance genes and the genes encoding transcription factors in response to Puccinia infection in wheat was the main objective of the present study.Methods: De novo assembly of SOLiD (sequencing by oligonucleotide ligation and detection) SAGE (serial analysis of gene expression) sequence reads from a pair of Near-isogenic lines (NILs) of wheat cultivar HD2329 with Lr28 (resistant) and HD2329 lacking Lr28 (susceptible) that were either infected with the most virulent pathogen Puccinia triticina or inoculated as mock in the absence of any reference sequence was carried out using multiple k-mer approach. Combinations of different software working on different algorithm were used to obtain a maximum number of differentially expressed transcripts.Results: De novo assembly at different k-mers produced a large number of contigs. The size of contigs was further increased with the use of different assembly software. Redundancy was removed both at nucleotide and protein levels, which increased the quality of assembly.Conclusion: For the assembly of short sequences of the complex genome such as those of polyploids a combination of software gives longer and unique contigs. It may be used in understanding the molecular mechanism of plant-microbe interaction.Keywords: Wheat, Leaf rust, SOLiD, SAGE, De novo assembly, NILs

DNA linkage based diagnosis of Wilson disease in asymptomatic siblings

Wilson disease (WD) is an autosomal recessive disorder caused by defects in ATP7B gene located in chromosome 13q14, and manifested as hepatolenticular degeneration as a result of accumulation of copper. No information on the mutation in the ATP7B gene and haplotypes using linked markers is available for WD patients in India. Hence, the present study was undetaken to identify, by a PCR-based molecular diagnostic test, presymptomatic siblings of WD affected individuals in families with multiple offspring. Methods: Genomic DNA was prepared from the peripheral blood of the patients, siblings and his/her first degree relatives. The repeat-markers flanking WD locus were amplified by PCR using fluorescent labeled primers. Amplified DNA fragments were analyzed by polyacrylamide gel electrophoresis in ABI 377 DNA sequencing system. Genotypes of the samples were determined using Genescan software. Haplotypes were determined based on segregation of the alleles in the families under study. Results: Among 15 WD affected families with multiple children, 4 cases were identified where younger siblings shared same genotype as the patient at all three markers analyzed. Further, eight different haplotypes were detected in the four patients. Interpretation & conclusion: The siblings of the WD patients carrying the same genotype at the markers linked to WD locus were presymptomatically diagnosed individuals. Presence of eight different haplotypes in the four patients suggested mutational heterogeneity at the WD locus. The test helps clinicians for therapeutic intervention in suspect WD cases by copper chelating agents prior to manifestation of overt clinical symptoms. Key words ATP7B - genotype - haplotype - microsatellite - Wilson disease (WD) is a genetic disorder, which manifests as hepatolenticular degeneration as a result of accumulation of copper in the brain, liver, kidney and cornea due to its deranged biliary excretion1. In 1912, a WD was described as a familial syndrome of progressive lenticular degeneration associated with cirrhosis of the liver2. The etiological role of copper in the pathogenesis of WD was recognized much late

ANTIBIOGRAM PROFILING OF HELICOBACTER PYLORI STRAINS AND THE EFFICACY OF BRASSICA CAPITATA AGAINST RESISTANT STRAINS ISOLATED FROM THE PATIENTS SUFFERING FROM GASTRODUODENAL DISEASES IN GUWAHATI, ASSAM

  Objective: Helicobacter pylori resistance toward commonly used antibiotics is increasing leading to the treatment failure; hence, our aim is to determine the antibiogram susceptibility pattern of H. pylori strains isolated from Guwahati, Assam (Northeast India) and also to test the efficacy of the Brassica capitata against the multi and dual drug-resistant strains of North and Northeast India.Methods: Minimum inhibitory concentration of different antibiotics was determined by agar dilution method. Disc diffusion method was used to check the efficacy of B. capitata against clarithromycin (CLR), metronidazole (MTZ), and levofloxacin (LEV)-resistant H. pylori strains.Results: All the H. pylori strains were 100% sensitive to CLR, tetracycline, amoxicillin, and furazolidone. 72.8% of the strains were sensitive toward MTZ and 54.5% were sensitive toward LEV. B. capitata showed good efficacy against the resistant strains of H. pylori of North and Northeast India.Conclusion: Most of the H. pylori strains from Northeast India were sensitive toward the commonly used antibiotics for the treatment regime. B. capitata is effective against H. pylori infection, suggesting its potential as an alternative therapy, and opens the way for further studies on identification of novel antimicrobial targets of B. capitata