Mahendra Kumar Modi

Computational identification and characterization of conserved miRNAs and their target genes in garlic (Allium sativum L.) expressed sequence tags.

The endogenous small non-coding functional microRNAs (miRNAs) are short in size, range from ~21 to 24 nucleotides in length, play a pivotal role in gene expression in plants and animals by silencing genes either by destructing or blocking of translation of homologous mRNA. Although various high-throughput, time consuming and expensive techniques like forward genetics and direct cloning are employed to detect miRNAs in plants but comparative genomics complemented with novel bioinformatic tools pave the way for efficient and cost-effective identification of miRNAs through homologous sequence search with previously known miRNAs. In this study, an attempt was made to identify and characterize conserved miRNAs in garlic expressed sequence tags (ESTs) through computational means. For identification of novel miRNAs in garlic, a total 3227 known mature miRNAs of plant kingdom Viridiplantae were searched for homology against 21,637 EST sequences resulting in identification of 6 potential miRNA candidates belonging to 6 different miRNA families. The psRNATarget server predicted 33 potential target genes and their probable functions for the six identified miRNA families in garlic. Most of the garlic miRNA target genes seem to encode transcription factors as well as genes involved in stress response, metabolism, plant growth and development. The results from the present study will shed more light on the understanding of molecular mechanisms of miRNA in garlic which may aid in the development of novel and precise techniques to understand some post-transcriptional gene silencing mechanism in response to stress tolerance.

In silico analyses of superoxide dismutases (SODs) of rice (Oryza sativa L.)

Superoxide dismutases (SODs), members of the metalloenzymes family are most effective intracellular enzymatic antioxidant in aerobic organisms. These enzymes provide the first line of defense in plants against the toxic effects of elevated levels of reactive oxygen species (ROS) generated during various environmental stresses. The availability of high-throughput computational tools has provided better opportunities to characterize the protein features and determine their function. In the present study an attempt was made to gain an insight into the structure and evolution of subunits of SODs (Cu-Zn, Mn and Fe SODs) of rice. The 3-Dimensional structures of SODs were modeled based on available X-ray crystal structures and further validated. The primary sequence, secondary and tertiary structure analysis revealed Mn and Fe SOD to be structurally homologous while Cu-Zn SOD is un-related to either of them. Comparative structural study also revealed former two were dominated by α-helices followed by β-strands in contrast; Cu-Zn SOD dominated by β-strands. Molecular phylogeny indicated a common evolutionary origin of Mn and Fe SOD while Cu-Zn SOD may have evolved separately.

Molecular cloning, characterization and expression analysis of 3-hydroxy-3-methylglutaryl coenzyme A reductase gene from Centella asiatica L.

Abstract3-Hydroxy-3-methylglutaryl-CoA reductases (HMGR) plays an important role in catalyzing the first committed step of isoprenoid biosynthesis in the mevelonic (MVA) pathway (catalyzes the conversion of HMG-CoA to MVA) in plants. The present manuscript reports the full length cDNA cloning of HMGR (CaHMGR, GenBank accession number: KJ939450.2) and its characterization from Centella asiatica. Sequence analysis indicated that the cDNA was of 1965xa0bp, which had an open reading frame of 1617xa0bp and encoded a protein containing 539 amino-acids with a molxa0wt of 57.9xa0kDa. A BLASTp search against non-redundant (nr) protein sequence showed that C. asiatica HMGR (CaHMGR) has 65–81xa0% identity with HMGRs from different plant species and multi-alignment comparison analysis showed the presence of two motif each corresponding to HMG-CoA-binding and NADP(H)-binding. The Conserved Domain Database analysis predicted that CaHMGR belongs to Class I hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase. Three-dimensional modeling confirmed the novelty of CaHMGR with a spatial structure similar to Homo sapiens (PDB id: 1IDQ8_A). Tissue Expression analysis indicates that CaHMGR is ubiquitous albeit differentially expressed among different tissues analysed, Strong expression was recorded in the nodes and leaves and low in the roots. The present investigation confirmed that nodes are vital to terpenoid synthesis in C. asiatica. Thus, the cloning of full length CDS, characterization and structure–function analysis of HMGR gene in Centella facilitate to understand the HMGR’s functions and regulatory mechanisms involved in mevalonate pathway in C. asiatica at genetic level.

Molecular recognition of avirulence protein (avrxa5) by eukaryotic transcription factor xa5 of rice (Oryza sativa L.): Insights from molecular dynamics simulations

The avirulence gene avrxa5 of bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) recognized by the resistant rice lines having corresponding resistance (xa5) gene in a gene-for-gene manner. We used a combinatorial approach involving protein-protein docking, molecular dynamics (MD) simulations and binding free energy calculations to gain novel insights into the gene-for-gene mechanism that governs the direct interaction of R-Avr protein. From the best three binding poses predicted by molecular docking, MD simulations were performed to explore the dynamic binding mechanism of xa5 and avrxa5. Molecular Mechanics/Poisson Boltzmann Surface Area (MM/PBSA) techniques were employed to calculate the binding free energy and to uncover the thriving force behind the molecular recognition of avrxa5 by eukaryotic transcription factor xa5. Binding free energy analysis revealed van der Waals term as the most constructive component that favors the xa5 and avrxa5 interaction. In addition, hydrogen bonds (H-bonds) and essential electrostatic interactions analysis highlighted amino acid residues Lys54/Asp870, Lys56/Ala868, Lys56/Ala866, Lys56/Glu871, Ile59/His862, Gly61/Phe858, His62/Arg841, His62/Leu856, Ser101/Ala872 and Ser105/Asp870 plays pivotal role for the energetically stability of the R-Avr complex. Insights gained from the present study are expected to unveil the molecular mechanisms that define the transcriptional activator mediated transcriptome modification in host plants.

Structure-Based Computational Study of Two Disease Resistance Gene Homologues (Hm1 and Hm2) in Maize (Zea mays L.) with Implications in Plant-Pathogen Interactions

The NADPH-dependent HC-toxin reductases (HCTR1 and 2) encoded by enzymatic class of disease resistance homologous genes (Hm1 and Hm2) protect maize by detoxifying a cyclic tetrapeptide, HC-toxin, secreted by the fungus Cochliobolus carbonum race 1(CCR1). Unlike the other classes resistance (R) genes, HCTR-mediated disease resistance is an inimitable mechanism where the avirulence (Avr) component from CCR1 is not involved in toxin degradation. In this study, we attempted to decipher cofactor (NADPH) recognition and mode of HC-toxin binding to HCTRs through molecular docking, molecular dynamics (MD) simulations and binding free energy calculation methods. The rationality and the stability of docked complexes were validated by 30-ns MD simulation. The binding free energy decomposition of enzyme-cofactor complex was calculated to find the driving force behind cofactor recognition. The overall binding free energies of HCTR1-NADPH and HCTR2-NADPH were found to be −616.989 and −16.9749 kJ mol−1 respectively. The binding free energy decomposition revealed that the binding of NADPH to the HCTR1 is mainly governed by van der Waals and nonpolar interactions, whereas electrostatic terms play dominant role in stabilizing the binding mode between HCTR2 and NADPH. Further, docking analysis of HC-toxin with HCTR-NADPH complexes showed a distinct mode of binding and the complexes were stabilized by a strong network of hydrogen bond and hydrophobic interactions. This study is the first in silico attempt to unravel the biophysical and biochemical basis of cofactor recognition in enzymatic class of R genes in cereal crop maize.

Rediscovering Medicinal Plants' Potential with OMICS: Microsatellite Survey in Expressed Sequence Tags of Eleven Traditional Plants with Potent Antidiabetic Properties

Herbal medicines and traditionally used medicinal plants present an untapped potential for novel molecular target discovery using systems science and OMICS biotechnology driven strategies. Since up to 40% of the worlds poor people have no access to government health services, traditional and folk medicines are often the only therapeutics available to them. In this vein, North East (NE) India is recognized for its rich bioresources. As part of the Indo-Burma hotspot, it is regarded as an epicenter of biodiversity for several plants having myriad traditional uses, including medicinal use. However, the improvement of these valuable bioresources through molecular breeding strategies, for example, using genic microsatellites or Simple Sequence Repeats (SSRs) or Expressed Sequence Tags (ESTs)-derived SSRs has not been fully utilized in large scale to date. In this study, we identified a total of 47,700 microsatellites from 109,609 ESTs of 11 medicinal plants (pineapple, papaya, noyontara, bitter orange, bermuda brass, ratalu, barbados nut, mango, mulberry, lotus, and guduchi) having proven antidiabetic properties. A total of 58,159 primer pairs were designed for the non-redundant 8060 SSR-positive ESTs and putative functions were assigned to 4483 unique contigs. Among the identified microsatellites, excluding mononucleotide repeats, di-/trinucleotides are predominant, among which repeat motifs of AG/CT and AAG/CTT were most abundant. Similarity search of SSR containing ESTs and antidiabetic gene sequences revealed 11 microsatellites linked to antidiabetic genes in five plants. GO term enrichment analysis revealed a total of 80 enriched GO terms widely distributed in 53 biological processes, 17 molecular functions, and 10 cellular components associated with the 11 markers. The present study therefore provides concrete insights into the frequency and distribution of SSRs in important medicinal resources. The microsatellite markers reported here markedly add to the genetic stock for cross transferability in these plants and the literature on biomarkers and novel drug discovery for common chronic diseases such as diabetes.

A comparative proteomic approach to analyse structure, function and evolution of rice chitinases: a step towards increasing plant fungal resistance

Glycoside hydrolase family 19 chitinases (EC 3.2.1.14) widely distributed in plants, bacteria and viruses catalyse the hydrolysis of chitin and play a major role in plant defense mechanisms and development. Rice possesses several classes of chitinase, out of which a single structure of class I has been reported in PDB to date. In the present study an attempt was made to gain more insight into the structure, function and evolution of class I, II and IV chitinases of GH family 19 from rice. The three-dimensional structures of chitinases were modelled and validated based on available X-ray crystal structures. The structural study revealed that they are highly α-helical and bilobed in nature. These enzymes are single or multi domain and multi-functional in which chitin-binding domain (CBD) and catalytic domain (CatD) are present in class I and IV whereas class II lacks CBD. The CatD possesses a catalytic triad which is thought to be involved in catalytic process. Loop III, which is common in all three classes of chitinases, reflects that it may play a significant role in their function. Our study also confirms that the absence and presence of different loops in GH family 19 of rice may be responsible for various sized products. Molecular phylogeny revealed chitinases in monocotyledons and dicotyledons differed from each other forming two different clusters and may have evolved differentially. More structural study of this enzyme from different plants is required to enhance the knowledge of catalytic mechanism and substrate binding.

Construction of cDNA library and preliminary analysis of expressed sequence tags from tea plant (Camellia sinensis (L) O. Kuntze)

Tea is the most popular non-alcoholic and healthy beverage across the world. The understanding of the genetic organization and molecular biology of tea plant, which is very poorly understood at present, is required for quantum increase in productivity and efficient use of germplasm for either cultivation or breeding program. Single-pass sequencing of randomly selected cDNA clones is the most widely accepted technique for gene identification and cloning. In the present study, a good quality cDNA library was constructed and preliminary analysis of ESTs was carried out. The titers of unamplified and amplified libraries were 1.4 × 10(6)pfu/ml and 5.27 × 10(8)pfu/ml respectively. A total of 210 cDNA clones from the constructed cDNA library were sequenced and analyzed. A total of 84 high quality Expressed Sequence Tags (ESTs) were generated, among which 71 ESTs had significant homology with sequences in NCBI non-redundant protein database by BLAST X analysis. About 80% ESTs had poly (A) tail at 3 end indicating that the cDNAs were full length. The database-matched ESTs were classified into putative cellular roles, viz. energy-related category (corresponding to 20% of total BLAST X matched ESTs), Transcription (14.2%), protein synthesis (14.2%) cell growth and division (8.6%), cell structure (5.7%), signal transduction (5.7%), transporters (2.9%), disease and defenses (2.9%), secondary metabolism (2.9%) and gene regulation (2.9%). This study provides an overview of the mRNA expression profile and first hand information of gene sequence expressed in tender leaves and apical buds of tea plant.

Structural Comparison, Substrate Specificity, and Inhibitor Binding of AGPase Small Subunit from Monocot and Dicot: Present Insight and Future Potential

ADP-glucose pyrophosphorylase (AGPase) is the first rate limiting enzyme of starch biosynthesis pathway and has been exploited as the target for greater starch yield in several plants. The structure-function analysis and substrate binding specificity of AGPase have provided enormous potential for understanding the role of specific amino acid or motifs responsible for allosteric regulation and catalytic mechanisms, which facilitate the engineering of AGPases. We report the three-dimensional structure, substrate, and inhibitor binding specificity of AGPase small subunit from different monocot and dicot crop plants. Both monocot and dicot subunits were found to exploit similar interactions with the substrate and inhibitor molecule as in the case of their closest homologue potato tuber AGPase small subunit. Comparative sequence and structural analysis followed by molecular docking and electrostatic surface potential analysis reveal that rearrangements of secondary structure elements, substrate, and inhibitor binding residues are strongly conserved and follow common folding pattern and orientation within monocot and dicot displaying a similar mode of allosteric regulation and catalytic mechanism. The results from this study along with site-directed mutagenesis complemented by molecular dynamics simulation will shed more light on increasing the starch content of crop plants to ensure the food security worldwide.

Structural analysis and molecular dynamics simulations of novel δ-endotoxin Cry1Id from Bacillus thuringiensis to pave the way for development of novel fusion proteins against insect pests of crops.

The theoretical three-dimensional structure of a novel δ-endotoxin Cry1Id (81xa0kDa) belonging to Cry1I class, toxic to many of the lepidopteran pests has been investigated through comparative modeling. Molecular dynamics (MD) simulations was carried out to characterize its structural and dynamical features at 10 ns in explicit solvent using the GROMACS version 4.5.4. Finally the simulated model was validated by the SAVES, WHAT IF, MetaMQAP, ProQ, ModFOLD and MolProbity servers. Despite low sequence identity with its structural homologs, Cry1Id not only resembles the previously reported Cry structures but also shares the common five conserved blocks of amino acid residues. Although the domain II of Cry1Id superpose well with its closest structural homolog Cry8Ea1, variation of amino acids and length in the apical loop2 of domain II was observed. In this work, we have hypothesized that the variations in apical loop2 might be the sole factor for providing variable surface accessibility to Cry1Id protein that could be important in receptor recognition. MD simulation showed the proposed endotoxin retains its stable conformation in aqueous solution. The result from this study is expected to aid in the development hybrid Cry proteins and new potent fusion proteins with novel specificities against different insect pests for improved pest management of crop plants.