Department of Biological Sciences
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Item Expression and functional analysis of rice genes involved in reproductive development and stress response(World Scientific, 2007) Sharma, RitaThe rice genome sequenced and annotated by the IRGSP has identified 37,544 protein-coding genes. In an effort to identify genes encoding transcription factors and signal transduction components, more than 7,000 genes belonging to 87 classes have been used to prepare a local database. Detailed analysis of genes for plant hormone response, CDPKs, C2H2 zinc-finger, and SET domain proteins unraveled interesting evolutionary aspects in relation to genes and the rice genome. A 51k microarray, SAGE analysis, and real-time polymerase chain reaction revealed differential expression of target genes during reproductive development and stress conditions. Several genes specific to reproductive floral organs and seed development have been identified. A large number of SAGE tags are observed from intergenic regions and antisense strands reflecting the unexplored transcription potential of the rice genome. Analysis of rice gene promoter activities has been undertaken in transgenic tobacco/Arabidopsis to demarcate regions conferring anther-/pollen-specific expression. OSISAP1, a gene coding for a stress-associated zinc-finger protein, and its promoter have been functionally validated in transgenic tobacco and rice. Genes for proteins interacting with OSISAP1 have also been found to be stress-inducible. Investigations on functional analysis of stress-responsive genes are in progress.Item Expression and functional analysis of rice genes involved in reproductive development and stress response(World Scientific, 2007) Sharma, RitaThe rice genome sequenced and annotated by the IRGSP has identified 37,544 protein-coding genes. In an effort to identify genes encoding transcription factors and signal transduction components, more than 7,000 genes belonging to 87 classes have been used to prepare a local database. Detailed analysis of genes for plant hormone response, CDPKs, C2H2 zinc-finger, and SET domain proteins unraveled interesting evolutionary aspects in relation to genes and the rice genome. A 51k microarray, SAGE analysis, and real-time polymerase chain reaction revealed differential expression of target genes during reproductive development and stress conditions. Several genes specific to reproductive floral organs and seed development have been identified. A large number of SAGE tags are observed from intergenic regions and antisense strands reflecting the unexplored transcription potential of the rice genome. Analysis of rice gene promoter activities has been undertaken in transgenic tobacco/Arabidopsis to demarcate regions conferring anther-/pollen-specific expression. OSISAP1, a gene coding for a stress-associated zinc-finger protein, and its promoter have been functionally validated in transgenic tobacco and rice. Genes for proteins interacting with OSISAP1 have also been found to be stress-inducible. Investigations on functional analysis of stress-responsive genes are in progresItem Expression analysis of calcium-dependent protein kinase gene family during reproductive development and abiotic stress conditions in rice (Oryza sativa L. ssp. indica)(Springer, 2007-07-18) Sharma, RitaCalcium-dependent protein kinases (CDPKs) are important sensors of Ca+2 flux in plants, which control plant development and responses by regulating downstream components of calcium signaling pathways. Availability of the whole genome sequence and microarray platform allows investigation of genome-wide organization and expression profile of CDPK genes in rice with a view to ultimately define their function in plant systems. Genome-wide analysis led to identification of 31 CDPK genes in rice after a thorough annotation exercise based upon HMM profiles. Twenty-nine already identified CDPK genes were verified and two new members were added to the CDPK gene family of rice. Relative expression of all these genes has been analyzed by using Affymetrix rice genome array™ during three vegetative stages, six stages of panicle (P1–P6) and five stages of seed (S1–S5) development along with three abiotic stress conditions, viz. cold, salt and desiccation, given to seedling. Thirty-one CDPK genes were found to express in at least one of the experimental stages studied. Of these, transcripts for twenty three genes accumulated differentially during reproductive developmental stages; nine of them were preferentially up-regulated only in panicle, five were up-regulated in stages of panicles as well as seed development, whereas, expression of one gene was found to be specific to the S1 stage of seed development. Eight genes were found to be down-regulated during the panicle and seed developmental stages. Six CDPK genes were found to be induced while the expression of one gene was down-regulated under stress conditions. The differential expression of CDPK genes during reproductive development and stress is suggestive of their involvement in the underlying signal transduction pathways. Furthermore, up-regulation of common genes both during reproductive development as well as stress responses is indicative of common element between reproduction and stress.Item Genome-wide identification of C2H2 zinc-finger gene family in rice and their phylogeny and expression analysis(Springer, 2007-07-04) Sharma, RitaTranscription factors regulate gene expression in response to various external and internal cues by activating or suppressing downstream genes in a pathway. In this study, we provide a complete overview of the genes encoding C2H2 zinc-finger transcription factors in rice, describing the gene structure, gene expression, genome localization, and phylogenetic relationship of each member. The genome of Oryza sativa codes for 189 C2H2 zinc-finger transcription factors, which possess two main types of zincfingers (named C and Q). The Q-type zinc fingers contain a conserved motif, QALGGH, and are plant specific, whereas C type zinc fingers are found in other organisms as well. A genome-wide microarray based gene expression analysis involving 14 stages of vegetative and reproductive development along with 3 stress conditions has revealed that C2H2 gene family in indica rice could be involved during all the stages of reproductive development from panicle initiation till seed maturation.Item Rice cytosine DNA methyltransferases – gene expression profiling during reproductive development and abiotic stress(Wiley, 2009-10-08) Sharma, RitaDNA methylation affects important developmental processes in both plants and animals. The process of methylation of cytosines at C-5 is catalyzed by DNA methyltransferases (MTases), which are highly conserved, both structurally and functionally, in eukaryotes. In this study, we identified and characterized cytosine DNA MTase genes that are activated with the onset of reproductive development in rice. The rice genome (Oryza sativa L. subsp. japonica) encodes a total of 10 genes that contain the highly conserved MTase catalytic domain. These genes have been categorized into subfamilies on the basis of phylogenetic relationships. A microarray-based gene expression profile of all 10 MTases during 22 stages ⁄ tissues that included 14 stages of reproductive development and five vegetative tissues together with three stresses, cold, salt and dehydration stress, revealed specific windows of MTase activity during panicle and seed development. The expression of six methylases was specifically ⁄ preferentially upregulated with the initiation of floral organs. Significantly, one of the MTases was also activated in young seedlings in response to cold and salt stress. The molecular studies presented here suggest a greater role for these proteins and the epigenetic process in affecting genome activity during reproductive development and stress than was previously anticipated.Item Comparative transcript profiling of TCP family genes provide insight into gene functions and diversification in rice and Arabidopsis(Academy Journals, 2010) Sharma, RitaPlant-specific TCP transcription factor family has been implicated in diverse aspects of growth and development. Rice and Arabidopsis genomes encode 26 and 24 TCP family genes, respectively. In this study, we have performed an inclusive analysis of their expression during 21 and 18 stages of development in rice and Arabidopsis, respectively. The assorted patterns of expression, exhibited by TCP family genes, provide an evidence for spatiotemporal regulation of their relative abundance throughout plant development. Further profiling of rice genes in three sub-stages of early panicle development revealed differential accumulation of nine genes during panicle initiation and organ development. QPCR-based expression profiling of selected rice genes, during four stages of anther, suggested their involvement in early anther development as well. Eleven genes of rice and seven of Arabidopsis were differentially expressed in response to three abiotic stress treatments viz., cold, dehydration and salt. In silico analysis of 5' regulatory regions of differentially expressed genes revealed the presence of previously characterized cis-regulatory elements. Duplications seem to have played major role in diversification of TCP family genes with 14 genes of rice and 10 of Arabidopsis lying on duplicated segments of the respective genomes. Most of the duplicated genes exhibited varied expression patterns. The knowledge obtained in this study will be useful for selection and assessment of the functions of individual genes using reverse genetics approaches.Item Modulation of transcription factor and metabolic pathway genes in response to water-deficit stress in rice(Springer, 2010-09-07) Sharma, RitaWater-deficit stress is detrimental for rice growth, development, and yield. Transcriptome analysis of 1-week-old rice (Oryza sativa L. var. IR64) seedling under water-deficit stress condition using Affymetrix 57 K GeneChip® has revealed 1,563 and 1,746 genes to be up- and downregulated, respectively. In an effort to amalgamate data across laboratories, we identified 5,611 differentially expressing genes under varying extrinsic water-deficit stress conditions in six vegetative and one reproductive stage of development in rice. Transcription factors (TFs) involved in ABA-dependent and ABA-independent pathways have been found to be upregulated during water-deficit stress. Members of zinc-finger TFs namely, C₂H₂, C₂C₂, C₃H, LIM, PHD, WRKY, ZF-HD, and ZIM, along with TF families like GeBP, jumonji, MBF1 and ULT express differentially under water-deficit conditions. NAC (NAM, ATAF and CUC) TF family emerges to be a potential key regulator of multiple abiotic stresses. Among the 12 TF genes that are co-upregulated under water-deficit, salt and cold stress conditions, five belong to the NAC TF family. We identified water-deficit stress-responsive genes encoding key enzymes involved in biosynthesis of osmoprotectants like polyols and sugars; amino acid and quaternary ammonium compounds; cell wall loosening and structural components; cholesterol and very long chain fatty acid; cytokinin and secondary metabolites. Comparison of genes responsive to water-deficit stress conditions with genes preferentially expressed during panicle and seed development revealed a significant overlap of transcriptome alteration and pathways.Item Microarray Analysis and Biochemical Correlations of Oxidative Stress Responsive Genes in Retinoblastoma(Taylor & Francis, 2012) Deepa, P.R.Oxidative stress, which refers to the biological damage caused by free radicals produced in excess of innate antioxidant defenses, is indicated in the ocular cancer retinoblastoma (RB). Here we have analysed the differential expression of oxidative stress responsive genes in oxidant-induced RB cells, and in RB tumor tissues.Item Design, construction and validation of a Plasmodium vivax microarray for the transcriptome profiling of clinical isolates(Elsiever, 2016-12) Das, Ashis KumarHigh density oligonucleotide microarrays have been used on Plasmodium vivax field isolates to estimate whole genome expression. However, no microarray platform has been experimentally optimized for studying the transcriptome of field isolates. In the present study, we adopted both bioinformatics and experimental testing approaches to select best optimized probes suitable for detecting parasite transcripts from field samples and included them in designing a custom 15K P. vivax microarray. This microarray has long oligonucleotide probes (60 mer) that were in-situ synthesized onto glass slides using Agilent SurePrint technology and has been developed into an 8X15K format (8 identical arrays on a single slide). Probes in this array were experimentally validated and represents 4180 P. vivax genes in sense orientation, of which 1219 genes have also probes in antisense orientation. Validation of the 15K array by using field samples (n = 14) has shown 99% of parasite transcript detection from any of the samples. Correlation analysis between duplicate probes (n = 85) present in the arrays showed perfect correlation (r2 = 0.98) indicating the reproducibility. Multiple probes representing the same gene exhibited similar kind of expression pattern across the samples (positive correlation, r ≥ 0.6). Comparison of hybridization data with the previous studies and quantitative real-time PCR experiments were performed to highlight the microarray validation procedure. This array is unique in its design, and results indicate that the array is sensitive and reproducible. Hence, this microarray could be a valuable functional genomics tool to generate reliable expression data from P. vivax field isolates.