Botany - Research Publications
Permanent URI for this collectionhttps://kr.cup.edu.in/handle/32116/32
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Item An overview of designing and selection of sgRNAs for precise genome editing by the CRISPR-Cas9 system in plants(Springer, 2019) Uniyal, A.P; Mansotra, K; Yadav, S.K; Kumar, VinayA large number of computational tools have been documented in recent years for identification of target-specific valid single-guide (sg) RNAs (18–20 nucleotide long sequence) that is an important component for the efficient utilization of the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats—CRISPR-associated Protein) system. Despite optimization of Cas9, other major concerns are on-target efficiency and off-target activity that depend upon the sequence(s) of target-specific sgRNA(s). However, a very little attention has been paid for identification of the best-hit sgRNA for precise targeting as well as minimizing the off-target effects. The aim of this present work is to offer comparative insight into existing CRISPR software tools with their unique features (including targeted genome) and utilities. These available web tools were found to be designed based upon only a few limited mathematical models. Among all these available web tools, three (Benchling, Desktop and CRISPR-P) have been curated as exclusively available for plant genome-editing purpose. These three software tools have been comprehensively described and analyzed with single same target enquiry from two randomly selected genes (IDM2 and IDM3 from Arabidopsis thaliana). Interestingly, all these selected tools generated different results (sgRNAs) even for the same query. In fact, the sequence of sgRNA is considered an important parameter to determine the efficiency and specificity of sgRNAs for precise genome editing. Thus, there is an urgent requirement to pay attention for a validated sgRNA-designing tool for precise DNA editing in plants. In conclusion, this work will encourage building up a consensus for developing a universal valid sgRNA designing for different organisms including plants. © 2019, King Abdulaziz City for Science and Technology.Item The CRISPR–Cas9, genome editing approach: a promising tool for drafting defense strategy against begomoviruses including cotton leaf curl viruses(Springer, 2019) Uniyal, A.P; Yadav, S.K; Kumar, VinayThe CRISPR–Cas9 is emerging genome editing tool and very easy and straightforward in operation that has been tested and explored for introduction of new traits in plant systems. Recently, a number of reports have documented utilization of this technology for providing tolerance against viral diseases mediated by begomoviruses. Begomoviruses infect dicot and are transmitted by white flies and cause devastating losses to yield of important agricultural crops including tomato, cassava and cotton. An overview of genomic structure of begomoviruses has been presented to understand the potential strategy for designing of effective sgRNAs to combat the viral replication for generating resistance against infection. This review provides the introduction, recent developments, and applications of the CRISPR–Cas9 system in plants and proposes a holistic methodology for generating cotton plant an example having resistance against begomoviruses. The genome editing using CRISPR–Cas9 system against complex of begomoviruses collectively termed as cotton leaf curl virus, which a major contributor to reduction of the cotton yield especially in Northern India and Pakistan is also discussed thoroughly. In conclusion, this potential strategy could be a sustainable approach for development of tolerant crops against diseases mediated by DNA viruses. © 2019, Society for Plant Biochemistry and Biotechnology.Item Current Approaches and Key Applications of Plant Metabolic Engineering(Springer, 2018) Bharti; Reetu; Kumar, VinayThe diversion of carbon flux toward biosynthesis of targeted products could be achieved by manipulation of targeted biosynthesis pathway in plants. This whole process consists of many steps in stepwise manners starting with the identification and isolation of targeted metabolites, elucidation of complete biosynthetic pathway for identification of point of intervention, discovery of corresponding potential metabolic genes, and overexpression of the selected genes in heterologous system and collectively production of the metabolites. The various biochemical processes including transcriptome, translatome, proteome, and reactome are being used to assist metabolic engineering by providing new insights into novel pathways or bottlenecks of existing pathways. Apart from all these, in-depth understanding of metabolic fluxes and feedback regulations is also mandatory for plant metabolic engineering. All these different current approaches are collectively considered for investigating the plant metabolic engineering to understand, reconstruct, analyze, and annotate the targeted pathways. The key applications of plant metabolic engineering have been compiled with a few important applications including improvement of nitrogen utilization in plant, development of highly nutritive food, and generation of biofuel production. In conclusion, the plant metabolic engineering could provide comprehensive evaluation of manipulation of biosynthetic pathways for numerous applications. This compiled information could act as a resource for crop breeding and biotechnology purposes.Item Factors defining the effects of macromolecular crowding on dynamics and thermodynamic stability of heme proteins in-vitro,(Elsevier, 2018) Kumar, Rajesh; Sharma, Deepak; Kumar, Vinay; Kumar, RajeshThe role of crowding agents on structure and activities of heme proteins has been established. Analysis of kinetic and thermodynamic parameters measured for CO-dissociation reaction of natively-folded carbonmonoxycytochrome c (NCO) and carbonmonoxymyoglobin (MbCO) at different [GdnHCl] or [Urea] in the presence of crowding agents (dextran 40, dextran 70 and ficoll 70) demonstrate that (i) at low denaturant concentrations, crowder presence enhances the denaturant-mediated restricted dynamics of NCO and MbCO, and (ii) at higher denaturant concentrations, large scale unfolding-fluctuations dominate the dynamics and inclusion of crowder counteracts the structural-fluctuations causing the unfolding of proteins. Thermodynamic analysis of thermal and urea-unfolding curves of cytochrome c (Cyt c) and myoglobin (Mb) measured at different [GdnHCl] in presence of crowding agents reveals that crowder presence counterbalances and strengthens the destabilizing action of GdnHCl on stability of Cyt c and Mb, respectively. This study further demonstrates that the size, shape and concentration of crowding agent modulate the effect of crowder on denaturant-mediated dynamics and thermodynamic stability of heme proteins.Item Macromolecular crowding-induced molten globule states of the alkali pH-denatured proteins(Elsevier B.V., 2018) Kumar, Rajesh; Kumar, Rajesh; Sharma, Deepak; Garg, Mansi; Kumar, Vinay; Agarwal, Mukesh ChandStructural and molecular properties extracted from circular dichroism (CD), tryptophan fluorescence and 1-anilino-8-napthalene sulfonate (ANS) binding experiments suggest that the high concentration of synthetic crowding agents (dextran 40, dextran 70 and ficoll 70) stabilizes and refolds the base-denatured ferricytochrome c (Ferricyt c) and lysozyme (Lyz) at pH 12.9 (±0.1) to molten globule (MG) states (C B -states). These results further revealed that the C B -states resemble the generic properties of MG-states. Thermodynamic analysis of thermal denaturation curves of base-denatured Ferricyt c and Lyz at pH 12.9 (±0.1) under variable concentrations of crowding agents (dextran 40, dextran 70 and ficoll 70) revealed that the crowder presence increases the thermal stability of base-denatured proteins and also prevents the cold denaturation of Ferricyt c. The results further showed that the nature, size and shape of crowder influence the crowding-mediated increase in secondary structure stabilization and thermal stability of base-denatured Ferricyt c and Lyz. Analysis of kinetic and thermodynamic parameters measured for CO association reaction of alkaline ferrocytochrome c (Ferrocyt c) at pH 12.9 (±0.1) under variable concentrations of crowding agents (dextran 40, dextran 70 and ficoll 70) revealed that the crowder presence reduces the level of structural fluctuation of M80-containing ?-loop that control CO association to alkaline Ferrocyt c. - 2018 Elsevier B.V.Item Pyramiding of tea Dihydroflavonol reductase and Anthocyanidin reductase increases flavan-3-ols and improves protective ability under stress conditions in tobacco(Springer Verlag, 2017) Kumar, Vinay; Yadav, Sudesh KumarTea (Camellia sinensis) is one of the richest sources of flavan-3-ols, an important class of flavonoids. The expression level of gene-encoded key regulatory enzymes of flavan-3-ol/anthocyanin biosynthetic pathway, dihydroflavonol 4-reductase (DFR) and anthocyanidin reductase (ANR), has been highly correlated with the flavan-3-ol contents and antioxidant activity in tea plant. In the present study, pyramiding of CsDFR and CsANR in tobacco was achieved. However, single transgenic tobacco overexpressing either CsDFR or CsANR was documented earlier. In continuation, pyramided transgenic lines were evaluated for the possible, either same or beyond, effect on flavan-3-ol accumulation and protective ability against biotic and abiotic stresses. The pyramided transgenic lines showed early flowering and improved seed yield. The transcript levels of flavan-3-ol/anthocyanin biosynthetic pathway and related genes in pyramided transgenic lines were upregulated as compared to control tobacco plants. The accumulations of flavan-3-ols were also found to be higher in pyramided transgenic lines than control tobacco plants. In contrast, anthocyanin content was observed to be decreased in pyramided transgenic lines, while DPPH activity was higher in pyramided transgenic lines. In pyramided transgenic lines, strong protective ability against feeding by Spodoptera litura was documented. The seeds of pyramided transgenic lines were also found to have better germination rate under aluminum toxicity as compared to control tobacco plants. Interestingly, the synergistic effect of these two selected genes are not beyond from transgenic lines expressing either CsDFR and CsANR alone as published earlier in terms of flavan-3-ols accumulation. However, the unique flower color and better seed germination rate are some interestingly comparable differences that were reported in pyramided lines in relation to individual transgenic plants. In conclusion, the present results reveal an interesting dynamic between CsDFR and CsANR in modulating flavan-3-ol/anthocyanin levels and functional analysis of stacked CsDFR and CsANR transgenic tobacco lines. ? 2017, Springer-Verlag GmbH Germany.Item Global transcriptome and coexpression network analyses reveal cultivar-specific molecular signatures associated with seed development and seed size/weight determination in chickpea.(Wiley, 2017) Garg, Rohini; Singh, Vikash K.; Rajkumar, Mohan Singh; Kumar, Vinay; Jain, MukeshSeed development is an intricate process regulated via a complex transcriptional regulatory network. To understand the molecular mechanisms governing seed development and seed size/weight in chickpea, we performed a comprehensive analysis of transcriptome dynamics during seed development in two cultivars with contrasting seed size/weight (small-seeded, Himchana 1 and large-seeded, JGK 3). Our analysis identified stage-specific expression for a significant proportion (>13%) of the genes in each cultivar. About one half of the total genes exhibited significant differential expression in JGK 3 as compared with Himchana 1. We found that different seed development stages can be delineated by modules of coexpressed genes. A comparative analysis revealed differential developmental stage specificity of some modules between the two cultivars. Furthermore, we constructed transcriptional regulatory networks and identified key components determining seed size/weight. The results suggested that extended period of cell division during embryogenesis and higher level of endoreduplication along with more accumulation of storage compounds during maturation determine large seed size/weight. Further, we identified quantitative trait loci-associated candidate genes harboring single nucleotide polymorphisms in the promoter sequences that differentiate small- and large-seeded chickpea cultivars. The results provide a valuable resource to dissect the role of candidate genes governing seed development and seed size/weight in chickpea.Item Anthocyanins enriched purple tea exhibits antioxidant, immunostimulatory and anticancer activities(Springer India, 2017) Joshi, Robin; Rana, Ajay; Kumar, Vinay; Kumar, Dharmesh; Padwad, Yogendra S.; Yadav, Sudesh Kumar; Gulati, AshuPurple coloured tea shoot clones have gained interest due to high content of anthocyanins in addition to catechins. Transcript expression of genes encoding anthocyanidin reductase (ANR), dihydroflavonol-4-reductase (DFR), anthocyanidin synthase (ANS), flavonol synthase (FLS) and leucoantho cyanidin reductase (LAR) enzymes in three new purple shoot tea clones compared with normal tea clone showed higher expression of CsDFR, CsANR, CsANS and lower expression of CsFLS and CsLAR in purple shoot clones compared to normal clone. Expression pattern supported high content of anthocyanins in purple tea. Four anthocyanins (AN1?4) were isolated and characterized by UPLC-ESI-QToF-MS/MS from IHBT 269 clone which recorded highest total anthocyanins content. Cyanidin-3-O-?-d-(6-(E)-coumaroyl) glucopyranoside (AN2) showed highest in vitro antioxidant activity (IC50?DPPH?=?25.27???0.02??g/mL and IC50?ABTS?=?10.71???0.01??g/mL). Anticancer and immunostimulatory activities of cyanidin-3-glucoside (AN1), cyanidin-3-O-?-d-(6-(E)-coumaroyl) glucopyranoside (AN2), delphinidin-3-O-?-d-(6-(E)-coumaroyl) glucopyranoside (AN3), cyanidin-3-O-(2-O-?-xylopyranosyl-6-O-acetyl)-?-glucopyranoside (AN4) and crude anthocyanin extract (AN5) showed high therapeutic perspective. Anthocyanins AN1?4 and crude extract AN5 showed cytotoxicity on C-6 cancer cells and high relative fluorescence units (RFU) at 200??g/mL suggesting promising apoptosis induction activity as well as influential immunostimulatory potential. Observations demonstrate potential of purple anthocyanins enriched tea clone for exploitation as a nutraceutical product. ? 2017, Association of Food Scientists & Technologists (India).