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ThesisItem Open Access Identification and networking of stress responsive gene families in maize(Department of Computational Biology and Bioinformatics Jacob Institute of Biotechnology and Bioengineering Sam Higginbottom University of Agriculture, Technology and Sciences (Formerly Allahabad Agricultural Institute-Deemed University) Allahabad- 211007, INDIA, 2019) MITTAL, SHIKHA; Jain, Dr. prashant AnkurDrought is one of the major threats to the maize yield especially in subtropical production systems. Understanding the genes and regulatory mechanisms of drought tolerance is important to sustain the yield. Transcription factors (TFs) and CDPK gene families play major role in gene regulation under drought stress. In the present study, a set of 15 major TF families comprising 1,436 genes and 273 CDPK proteins were structurally and functionally characterized. The functional annotation indicated that the genes were involved in ABA signaling, ROS scavenging, photosynthesis, stomatal regulation, and sucrose metabolism. Duplication was identified as the primary force in divergence and expansion of CDPK and TF families. Phylogenetic relationship was developed for individual TF and combined TF families and CDPK. Phylogenetic analysis clustered the genes into specific and mixed groups. In CDPK, four groups were identified from the sequence-based phylogenetic analysis, in which maize CDPKs were clustered in group III. Gene structure analysis revealed that more number of genes were intron-rich as compared to intron-less. Drought-responsive cis-regulatory elements such as ABREA, ABREB, DRE1, and DRECRTCOREAT have been identified. Expression and interaction analyses identified leaf-specific bZIP TF, GRMZM2G140355, as a potential contributor toward drought tolerance in maize. Protein-protein interaction network of 269 drought-responsive genes belonging to different TFs has been provided. The information generated on structural and functional characteristics, expression, and interaction of the drought-related TF families will be useful to decipher the drought tolerance mechanisms and to breed drought-tolerant genotypes in maize. Expression data showed that the CDPK genes were highly expressed in leaf of maize, rice, and sorghum whereas in Arabidopsis the maximum expression was observed in root. The expression assay showed 5, 6, 11, and 9 were the commonly and differentially expressed drought-related orthologous genes in maize, Arabidopsis, rice, and sorghum, respectively. 3-D protein structure were predicted for the nine genes (Arabidopsis: 2, maize: 2, rice: 3, and sorghum: 2) showing differential expression in at least three species. The predicted 3-D structures were further evaluated and validated by Ramachandran plot, ANOLEA, ProSA, and Verify-3D. The superimposed 3-D structure of drought-related orthologous proteins retained similar folding pattern owing to their conserved nature. Functional annotation revealed the involvement of CDPK genes in various pathways such as osmotic homeostasis, cell protection, and root growth. The interactions of CDPK genes in various pathways play crucial role in imparting drought tolerance through different ABA and MAPK signaling cascades. These selected candidate genes could be targeted in development of drought tolerant genotypes in maize, rice, and sorghum through appropriate breeding approaches. Our comparative experiments of CDPK genes could also be extended in the drought stress breeding programmes of the related species.ThesisItem Open Access Computational approach for identification of biotic stresses responsive genes and the host-pathogen interaction study of deciphered R genes (Ve1 and Cf4) in Solanum lycopersicum(Department of Computational Biology and Bioinformatics Jacob Institute of Biotechnology and Bioengineering Sam Higginbottom University of Agriculture, Technology and Sciences (Formerly Allahabad Agricultural Institute), Allahabad – 211007, U.P., INDIA, 2018) TANDON, GITANJALI; Singh, Dr. SatendraTomato is one of the major vegetable plant and a model system for fruit development. Its global importance is due to its lycopene pigment which has anti-oxidative and anti-cancerous properties. Being a tropical plant, it can very well adapt in almost all climatic conditions but still it is confronted by various environmental stresses which lead to the decrease of tomato production. These stresses can be either biotic or abiotic which causes the decrease in its production by 70% to 80%. Among these two, biotic stress causes the yield loss by 70-90%. Therefore, the present work mainly focuses on the identification of new genes related to biotic stress in tomato and annotation of the already predicted R genes (Ve1 and Cf-4) by carrying out there host pathogen interaction studies. For this > 1.5 M biotic stress associated ESTs of tomato are available but cumulative analysis to predict genes is warranted. Availability of whole genome de novo assembly can advantageously be used to map them over different chromosome. Total 55 novel genes associated with biotic stress of tomato along with 50 genes having physical location over different chromosomes have been reported in this work. 52 cis-regulating elements and 69 putative miRNAs which are involved in regulation of these biotic stresses associated genes have also been deciphered in the present work. These putative candidate genes associated with biotic stress can be used in molecular breeding in the endeavor of tomato productivity along with its sustainable germplasm management. From the annotated data set R-genes (Ve1 and Cf-4) were identified and subsequently their protein- protein interaction was studied with the effector proteins of the pathogen. Ve1 is the Rgene corresponding to the vascular wilt diseases caused by soil-borne pathogens Verticillium dahliae and Verticillium albo-atrum while Cf-4 confer resistance to the leaf mold disease caused by biotrophic fungus Cladosporium fulvum. During interaction analysis it was revealed that in Ve1 and Avr1 complex 35 favorable interactions were present while in case of Cf-4 and Avr4 complex 24 favorable interactions were available. Detailed insight of residues along with their atomic groups participating in the interaction have also been studied. This study helped in shifting the genomic knowledge to proteomic level and provides the better understanding of the diseases. It also provides the insight how proteins behave during the attack by the pathogens.ThesisItem Open Access Computational prediction of stress responsive genes and analysis of differentially expressed lncRNAs from Mesorhizobium inoculated genes of Cicer arietinum(Department of Computational Biology and Bioinformatics Jacob Institute of Biotechnology and Bioengineering Sam Higginbottom University of Agriculture, Technology,and Sciences (Formerly Allahabad Agricultural Institute), Allahabad- 211007, U.P., INDIA 2018, 2018) KAUR, SUKHDEEP; Singh, Dr. SatendraCicer arietinum (Chickpea) is genome of interest for many researchers, because genome of chickpea is recently decoded and so many unknown facts are to be explored and identified. Chickpea is an valuable crop, any damage to yield affect the global food supply. Many stresses and diseases are affecting chickpea at reproductive and growth stage. Objective of the study is prediction of gene, transcriptomic analysis to extract genetic information of stress-related genes and transcripts. In present study data from wide range of techniques; like expressed sequence tags, genomic survey sequences and sequencing reads were used. Functional characterization of uncharacterized chickpea genes has been carried out based on their homology with closely related model legume species Medicago truncatula for abiotic and biotic stresses like drought, salinity, cold and Ascochyta blight, Fusarium wilt. Other than stress and disease-related issues that adversely affect the yield of chickpea crop, a major physiological advantage occurs in root morphology of chickpea i.e. key phenomenon of nodulation. Nodulation in chickpea roots fixes the nitrogen for development and growth of the plant. Researchers are looking forward to more organic means of fertilizers than chemical-based fertilizers and symbiotic association of rhizobia and chickpea root in nodulation process are emerging to be one of them. Transcriptome and differential gene expression analysis was carried out in three stages of Mesorhizobium inoculated chickpea roots viz. early, middle and the late and control stage that leads to the identification of non-coding RNAs and their roles during symbiotic association. Two types of non-coding RNAs were identified; long non-coding RNAs (lncRNAs) and micro RNAs (miRNAs) Along with this SNP and SSR markers and their 5 primer pair sets, and transcription factor were also identified. A total of 682 lncRNAs were detected while 614 were common in all three stages. Specifically, 15, 14 and 29 lncRNAs were uniquely expressed in early, middle and late stages, respectively. lncRNAs that are expressed in all three stages are Ca_lncRNA_18179, Ca_lncRNA_25027, Ca_lncRNA_8484, Ca_lncRNA_9096. On analysis of Transcriptome and genome-wide distribution of identified miRNAs, total 15 miRNA/isomiRs found conserved across other species. Their target analysis confirms there role in nodulation process. All these findings, related stress genes and non-coding RNAs and genes during inoculation provides a great understanding and can add on in chickpea improvement programmes..ThesisItem Open Access “Identification of Unique anti- Atherosclerosis Inhibitors from Herbal plants – A Computational Approach”(DEPARTMENT OF COMPUTATIONAL BIOLOGY AND BIOINFORMATICS JACOB SCHOOL OF BIOTECHNOLOGY AND BIOENGINEERING SAM HIGGINBOTTOM INSTITUTE OF AGRICULTURE, TECHNOLOGY AND SCIENCES Deemed-to-be-University (FORMERLY ALLAHABAD AGRICULTURAL INSTITUTE) NAINI, ALLAHABAD, UP 211007, 2018) DUBEY, AMITThe human chymase (EC 3.4.21.39) is a serine protease abundant in secretory granules of mast cells, that possesses chymotrypsin-like cleavage specificity and is responsible and is involved in the synthesis of angiotensin II from its precursor. Moreover, it is also responsible for the activation and conversion of transforming growth factor-β (TGF-β) and matrix metalloproteinase (MMP)-9 precursors to their active forms. Chymase-induced activation of TGF-b and MMP-9 shows significance in cardiovascular disease. Since a link between atherosclerosis and chymase has been proved, specific chymase inhibitors are actively searched to develop new therapeutic treatments for cardiovascular diseases, considering also that chymase has no enzymatic activity in normal tissues. Therefore, specific chymase inhibitors may not have effects on any other target in normal tissues. The use of natural products has been an integral part of the treatment of different diseases throughout the world since past centuries. Many plants with potential therapeutic activity were widely used as natural medicines with negligible undesired effects. Therefore, the search for new active compounds from natural sources is gaining interest in the scientific community, and the application of novel approaches to an old science could result in the discovery of valuable compounds useful to develop innovative drugs. The aim of the present study was to search for novel inhibitors of chymase enzyme from natural sources or inspired by nature, using a computational approach that allowed us to screen databases of compounds and to predict which molecules can be able to bind to chymase with a good affinity and selectivity against other human serine proteases (kallikrein, elastase and tryptase). Among the selected compounds obtained from ZINC Biogenic database, ii “ZINC22061412 and ZINC04416136"are predicted to bind human chymase with a binding energy comparable, or even better, with respect to the one predicted for known inhibitors. Among the compounds selected from ginkgo biloba and chamomile plants, all show a negative binding energy, suggesting a potential ability to interact with the enzyme, but the absolute values are often higher with respect to those of known inhibitors, indicating a less favorable interaction. Combining previous computational studies, at the final stage were performed on natural compounds subsets from different nutraceutical sources, highlighting Density Functional Theory-based quantum mechanical studies on good candidate screened by generated pharmacophore models. Other electronic properties were calculated such as LUMO, HOMO, and locations of molecular electrostatic potentials, for identification of potent hits that can bind to various bioactive conformations available in the active site of enzyme. Finally four best candidate, Cynarine, Curcumin, Scutellarin, and Ginkgolic acid show a predicted binding energy equal or even better than those predicted for many known chymase inhibitors. The analysis of the complexes with the best predicted energies shows that these molecules can interact with the key residues involved in substrate binding and catalysis. Moreover, several compounds are able to interact with chymase with a good selectivity towards the other tested serine proteases, since the predicted binding energy for these last enzymes is in some cases significantly higher with respect to that for chymase. In conclusion, the selected molecules can become novel candidates for the therapeutic action in the context of cardiovascular diseases, making them good candidates for further experimental studies to characterize their activity in vitro and in vivo. Our results also shows a possible explanation of their inhibitory mechanism and important structural insights which extend my expertise for drug designing against enzyme targets, enhancing the possibility for the potential development of future drugs.ThesisItem Open Access Genome-wide identification and comprehensive analysis of Excretory/Secretory proteins in nematode provides potential targets for parasite control(DEPARTMENT OF COMPUTATIONAL BIOLOGY AND BIOINFORMATICS JACOB INSTITUTE OF BIOTECHNOLOGY AND BIOENGINEERING SAM HIGGINBOTTOM UNIVERSITY OF AGRICULTURE, TECHNOLOGY AND SCIENCES (FORMERLY ALLAHABAD AGRICULTURAL INSTITUTE-DEEMED UNIVERSITY), ALLAHABAD – 211 007, INDIA, 2017) GAHOI, SHACHI; Gautam, BudhayashNematodes are the most numerous multicellular animals on earth and causes severe diseases in plants, humans and other animals. Infection is associated with the secretion of effector proteins into host cytoplasm and interference with host innate immunity. To combat this infection, the identification and functional annotation of Excretory/Secretory (ES) proteins serve as a key to produce durable control measures. The identification of ES proteins through experimental methods are expensive and time consuming while bioinformatics approaches are cost- and time-effective by prioritizing the experimental analysis of potential drug targets for parasitic diseases. Using nextgeneration sequencing technology, several nematode genomes have been sequenced. In this study, total of 93,949 ES proteins were predicted and functionally annotated in the genome of 73 nematodes using integration of several bioinformatics tools. Interestingly, RNA support was found for 28,808 ES proteins representing 30.66% of the identified ES proteins. The functional analysis could annotate 50,099 (53.3%) of ES proteins to protein domains, 22,516 (24%) proteins using Gene Ontology (GO) terms, and established pathway associations for 10,227 (10.9%) sequences using Kyoto Encyclopaedia of Genes and Genomes (KEGG). Many important gene families, reported to be involved in parasitic activity such as C-type lectin, protein kinases and peptidases were found to be highly represented in identified ES protein set. The most represented GO terms in predicted ES proteins were catalytic activity, binding and hydrolase activity which are strongly associated with typical functions of secreted proteins. The metabolic pathway analyses identified several important pathways in ES proteins essential for parasite survival inside the host which include protein processing in endoplasmic reticulum, lysosome, pathways in cancer, PI3K-Akt signaling pathway, proteoglycans in cancer, HTLV-I infection and purine metabolism. Interestingly, the enzyme class analyses result showed an overrepresentation of hydrolases and oxidoreductases in the ES proteins as compared to the same enzyme classes for the non- ES proteins for all the genome of studied nematodes. Moreover, 11,312 (12%) of ES proteins were mapped to protease classes, 2972 (3.16%) to CAZyme families and 1610 (1.7%) of ES proteins to C. elegans 115 neuropeptide genes, belonged to ins, flp and nlp gene classes. The ins-1, ins-17 and ins-18 were the only ins families which were detected in all the studied nematode groups. The flp-1, flp-3, flp-6, flp-7, flp-8, flp-9, flp-13, flp-14, flp-17 and flp-18 were the prominent members identified in flp gene class whereas nlp-4 was found to be highly represented nlp gene class in secretome of all studied nematode groups. Additionally, 492 ES proteins were found to be homologues to DrugBank approved target database and C. elegans RNA interference gene database which could represent potential targets for parasite control. This comprehensive analysis of nematode secretome provides valuable resource for further experimental studies to understand host-pathogen interactions and novel therapeutic solutions for parasite infections.ThesisItem Open Access Identification of Unique anti- Atherosclerosis Inhibitors from Herbal plants – A Computational Approach(Sam Higginbottom Institute of Agriculture, Technology & Sciences (SHIATS), 2016) Dubey, Amit; Ramteke, Prof. (Dr.) Pramod WThe human chymase (EC 3.4.21.39) is a serine protease abundant in secretory granules of mast cells, that possesses chymotrypsin-like cleavage specificity and is responsible and is involved in the synthesis of angiotensin II from its precursor. Moreover, it is also responsible for the activation and conversion of transforming growth factor-β (TGF-β) and matrix metalloproteinase (MMP)-9 precursors to their active forms. Chymase-induced activation of TGF-b and MMP-9 shows significance in cardiovascular disease. Since a link between atherosclerosis and chymase has been proved, specific chymase inhibitors are actively searched to develop new therapeutic treatments for cardiovascular diseases, considering also that chymase has no enzymatic activity in normal tissues. Therefore, specific chymase inhibitors may not have effects on any other target in normal tissues. The use of natural products has been an integral part of the treatment of different diseases throughout the world since past centuries. Many plants with potential therapeutic activity were widely used as natural medicines with negligible undesired effects. Therefore, the search for new active compounds from natural sources is gaining interest in the scientific community, and the application of novel approaches to an old science could result in the discovery of valuable compounds useful to develop innovative drugs. The aim of the present study was to search for novel inhibitors of chymase enzyme from natural sources or inspired by nature, using a computational approach that allowed us to screen databases of compounds and to predict which molecules can be able to bind to chymase with a good affinity and selectivity against other human serine proteases (kallikrein, elastase and tryptase). Among the selected compounds obtained from ZINC Biogenic database, ii “ZINC22061412 and ZINC04416136"are predicted to bind human chymase with a binding energy comparable, or even better, with respect to the one predicted for known inhibitors. Among the compounds selected from ginkgo biloba and chamomile plants, all show a negative binding energy, suggesting a potential ability to interact with the enzyme, but the absolute values are often higher with respect to those of known inhibitors, indicating a less favorable interaction. Combining previous computational studies, at the final stage were performed on natural compounds subsets from different nutraceutical sources, highlighting Density Functional Theory-based quantum mechanical studies on good candidate screened by generated pharmacophore models. Other electronic properties were calculated such as LUMO, HOMO, and locations of molecular electrostatic potentials, for identification of potent hits that can bind to various bioactive conformations available in the active site of enzyme. Finally four best candidate, Cynarine, Curcumin, Scutellarin, and Ginkgolic acid show a predicted binding energy equal or even better than those predicted for many known chymase inhibitors. The analysis of the complexes with the best predicted energies shows that these molecules can interact with the key residues involved in substrate binding and catalysis. Moreover, several compounds are able to interact with chymase with a good selectivity towards the other tested serine proteases, since the predicted binding energy for these last enzymes is in some cases significantly higher with respect to that for chymase. In conclusion, the selected molecules can become novel candidates for the therapeutic action in the context of cardiovascular diseases, making them good candidates for further experimental studies to characterize their activity in vitro and in vivo. Our results also shows a possible explanation of their inhibitory mechanism and important structural insights which extend my expertise for drug designing against enzyme targets, enhancing the possibility for the potential development of future drugs.