Mousumi Sahu

In silico identification and characterization of conserved miRNAs and their target genes in sweet potato (Ipomoea batatas L.) Expressed Sequence Tags (ESTs)

The endogenous small non-coding micro RNAs (miRNAs), which are typically ~21–24 nt nucleotides, play a crucial role in regulating the intrinsic normal growth of cells and development of the plants as well as in maintaining the integrity of genomes. These small non-coding RNAs function as the universal specificity factors in post-transcriptional gene silencing. Discovering miRNAs, identifying their targets, and further inferring miRNA functions is a routine process to understand normal biological processes of miRNAs and their roles in the development of plants. Comparative genomics based approach using expressed sequence tags (EST) and genome survey sequences (GSS) offer a cost-effective platform for identification and characterization of miRNAs and their target genes in plants. Despite the fact that sweet potato (Ipomoea batatas L.) is an important staple food source for poor small farmers throughout the world, the role of miRNA in various developmental processes remains largely unknown. In this paper, we report the computational identification of miRNAs and their target genes in sweet potato from their ESTs. Using comparative genomics-based approach, 8 potential miRNA candidates belonging to miR168, miR2911, and miR156 families were identified from 23 406 ESTs in sweet potato. A total of 42 target genes were predicted and their probable functions were illustrated. Most of the newly identified miRNAs target transcription factors as well as genes involved in plant growth and development, signal transduction, metabolism, defense, and stress response. The identification of miRNAs and their targets is expected to accelerate the pace of miRNA discovery, leading to an improved understanding of the role of miRNA in development and physiology of sweet potato, as well as stress response.

Mining for SSRs and FDMs from expressed sequence tags of Camellia sinensis

Simple Sequence Repeats (SSRs) developed from Expressed Sequence Tags (ESTs), known as EST-SSRs are most widely used and potentially valuable source of gene based markers for their high levels of crosstaxon portability, rapid and less expensive development. The EST sequence information in the publicly available databases is increasing in a faster rate. The emerging computational approach provides a better alternative process of development of SSR markers from the ESTs than the conventional methods. In the present study, 12,851 EST sequences of Camellia sinensis, downloaded from National Center for Biotechnology Information (NCBI) were mined for the development of Microsatellites. 6148 (4779 singletons and 1369 contigs) non redundant EST sequences were found after preprocessing and assembly of these sequences using various computational tools. Out of total 3822.68 kb sequence examined, 1636 (26.61%) EST sequences containing 2371 SSRs were detected with a density of 1 SSR/1.61 kb leading to development of 245 primer pairs. These mined EST-SSR markers will help further in the study of variability, mapping, evolutionary relationship in Camellia sinensis. In addition, these developed SSRs can also be applied for various studies across species.

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.

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.

ESMP: A high-throughput computational pipeline for mining SSR markers from ESTs.

With the advent of high-throughput sequencing technology, sequences from many genomes are being deposited to public databases at a brisk rate. Open access to large amount of expressed sequence tag (EST) data in the public databases has provided a powerful platform for simple sequence repeat (SSR) development in species where sequence information is not available. SSRs are markers of choice for their high reproducibility, abundant polymorphism and high inter-specific transferability. The mining of SSRs from ESTs requires different high-throughput computational tools that need to be executed individually which are computationally intensive and time consuming. To reduce the time lag and to streamline the cumbersome process of SSR mining from ESTs, we have developed a user-friendly, web-based EST-SSR pipeline “EST-SSR-MARKER PIPELINE (ESMP)”. This pipeline integrates EST pre-processing, clustering, assembly and subsequently mining of SSRs from assembled EST sequences. The mining of SSRs from ESTs provides valuable information on the abundance of SSRs in ESTs and will facilitate the development of markers for genetic analysis and related applications such as marker-assisted breeding. Availability The database is available for free at http://bioinfo.aau.ac.in/ESMP

Molecular Phylogeny, Homology Modeling, and Molecular Dynamics Simulation of Race-Specific Bacterial Blight Disease Resistance Protein (xa5) of Rice: A Comparative Agriproteomics Approach

Rice (Oryza sativa L.), a model plant belonging to the family Poaceae, is a staple food for a majority of the people worldwide. Grown in the tropical and subtropical regions of the world, this important cereal crop is under constant and serious threat from both biotic and abiotic stresses. Among the biotic threats, Xanthomonas oryzae pv. oryzae, causing the damaging bacterial blight disease in rice, is a prominent pathogen. The xa5 gene in the host plant rice confers race-specific resistance to this pathogen. This recessive gene belongs to the Xa gene family of rice and encodes a gamma subunit of transcription factor IIA (TFIIAγ). In view of the importance of this gene in conferring resistance to the devastating disease, we reconstructed the phylogenetic relationship of this gene, developed a three-dimensional protein model, followed by long-term molecular dynamics simulation studies to gain a better understanding of the evolution, structure, and function of xa5. The modeled structure was found to fit well with the small subunit of TFIIA from human, suggesting that it may also act as a small subunit of TFIIA in rice. The model had a stable conformation in response to the atomic flexibility and interaction, when subjected to MD simulation at 20 nano second in aqueous solution. Further structural analysis of xa5 indicated that the protein retained its basic transcription factor function, suggesting that it might govern a novel pathway responsible for bacterial blight resistance. Future molecular docking studies of xa5 underway with its corresponding avirulence gene is expected to shed more direct light into plant-pathogen interactions at the molecular level and thus pave the way for richer agriproteomic insights.

An approach to delineate primers for a group of poorly conserved sequences incorporating the common motif region.

Glutathione synthetase (gshB) has previously been reported to confer tolerance to acidic soil condition in Rhizobium species. Cloning the gene coding for this enzyme necessitates the designing of proper primer sets which in turn depends on the identification of high quality sequence similarity in multiple global alignments. In this experiment, a group of homologous gene sequences related to gshB gene (accession no: gi-86355669:327589-328536) of Rhizobium etli CFN 42, were extracted from NCBI nucleotide sequence databases using BLASTN and were analyzed for designing degenerate primers. However, the T-coffee multiple global alignment results did not show any block of conserved region for the above sequence set to design the primers. Therefore, we attempted to identify the location of common motif region based on multiple local alignments employing the MEME algorithm supported with MAST and Primer3. The results revealed some common motif regions that enabled us to design the primer sets for related gshB gene sequences. The result will be validated in wet lab.