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2014 - 20152
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Subject: Biotechnology × End date: 2015 × Start date: 2010 × Type: Thesis ×
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    ThesisItemOpen Access
    Identification and characterization of viruses in sweet potato (Ipomoea batatas (L.) Lam.)
    (Department of Plant Biotechnology, College of Agriculture, Vellayani, 2015) Jayalekshmi, V S; KAU; Makeshkumar, T
    The study entitled "identification and characterization of viruses in sweet potato (Ipomoea batatas (L) Lam.) was carried out at the division of crop protection, Central Tuber Crops Research Institute, Sreekariyam, Trivandrum during 2014-2015. The objective of the study was to diagnose, clone and characterize viruses implicated in mixed infections of sweet potato. Sweet potato samples with various virus infection symptoms were collected from the germplasm repository of CTCRI, Trivandrum and field samples from Bhubaneswar. Samples were screened mainly for Sweet potato feathery mottle virus ( SPFMV ), Sweet potato mild mottle virus ( SPMMV), Sweet potato leaf curl virus (SPLCV ), Sweet potato chlorotic stunt virus ( SPCSV), Sweet potato virus G (SPVG), Sweet potato virus C (SPVC), Sweet potato virus 2 (SPV2) using both genus and virus specific primers. Out of 32, 29 samples showed SPFMV infection in PCR with virus specific primers. While mixed infection by SPFMV and SPLCV was found in 15 samples. One sample was infected with SPVG along with SPFMV and SPLCV. SPMMV, SPVC, SPV2 and SPCSV screening through PCR gave negative results for all samples. PCR by virus specific primers of SPFMV and SPLCV amplifying the partial CP gave amplicons size of 411 bp and 446 bp respectively. Rather than the virus specific primers, the group specific primers Pot1/Hrp5 gave an amplicon of 1300 bp lead to the detection of SPVG. After identification, one sample each for SPFMV, SPLCV and the only sample positive for SPVG were cloned and sequenced. The sequence data was analyzed through BLAST and sequence similarity was studied. The 304 nt SPFMV sequence obtained in the study showed maximum similarity of 96% to Sweet potato feathery mottle virus isolate Fe polyprotein gene, partial cds (Accession EU021070). The 251 nt SPVG sequence obtained showed maximum similarity of 90% to sweet potato virus G isolate IS103, complete genome (AccessionKM014815). While the 418 nt SPLCV sequence obtained showed maximum similarity of 96% to Sweet potato leaf curl virus DNA A, complete sequence (Accession AF104036) and Sweet potato leaf curl isolate CTCRI TVM M1, complete genome (Accession KM 050768). The phylogenetic tree was constructed with similar sequences using phylip. Phylogenetic analysis clearly revealed that the sequences obtained in this study belongs to SPFMV for the sample S1294, SPLCV for the sample S1294, SPLCV for the sample S684 and SPVG for the sample S270 as they grouped along with their respective virus sequences used for comparison analysis. Since the diagnosis of virus infections based on symptoms is unreliable due to complicated mixed infections in sweet potato with multiple viruses and isolates, it is necessary sensitive diagnostic tests are developed region wise to confront this issue. As a prerequisite to this, virus detection and identification has to be carried out in sweet potato to determine the viruses geographically.
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    ThesisItemOpen Access
    Identification of lead compounds with anti-tuberculosis activity in indegenous spices of Kerala
    (College of Agriculture, Vellayani, 2014) Arun, Jyothi P V; KAU; Sreekumar, S
    Tuberculosis (TB) caused by Mycobacterium tuberculosis is the second worldwide killer infectious disease and it kills annually 1.4 million people globally and 30,000 people in India. Although drugs are available to treat tuberculosis they have several limitations including long term treatment, side effects, emergence of multidrug-resistant (MDR) and extensively drug resistant (XDR) mutants and adverse effect to immune system in patients co-infection with HIV. Therefore, discovery of novel faster, cheaper and better drug is become the need of the hour. Since time immemorial several herbal remedies have been used against tuberculosis in the traditional systems of treatment especially in India and in African countries. The indigenous spices of Kerala are well known for its use to treat human respiratory system. But its efficacies and mode of action are seldom investigated. In the present study the phytomolecules reported from Elettaria cardamomum, Curcuma longa and Zingiber officinale were screened through in silico and in vitro methods. For in silico screening Decaprenylphosphoryl-beta-D-ribose epimerase (DprE1), an enzyme responsible for the synthesis of arabinan, the virulent factor in M., tuberculosis was selected as the target molecule. The 3-D structure of the molecule was retrieved from PDB (PDB id 4FDO). The active site DprE1 was detected using the tool PDBsum. Information regarding the chemical molecules reported in the selected spices was collected through literature survey and databases. The canonical SMILES of the phytochemicals were retrieved from open access chemical databases and 3D structures were created using CORINA. Total 448 phytochemicals (C. longa – 211, Z. officinale – 183 and ¬E. cardamomum – 54) were screened. Out of 448 phytochemicals structures of 373 (C. longa –137, Z. officinale –182 and ¬E. cardamomum –0) were retrieved from databases and remaining compound’s structures were created using Chemsketch. All selected phytochemicals were docked into the binding site of DprE1 using the tool, AutoDock 4.2. The docked structures having ΔG less than -5 kcal mol-1 were selected as best hit molecules. Out of 211 compounds screened in C. longa 101, out of 183 compounds screened in Z. officinale 63 and out of 54 compounds screened in E. cardamomum 22 of them showed free energy of binding  -5 kcal mol-1 and these molecules were further analysed by Lipinski's rule of Five. To nullify the errors in lead identification the top ranked hit molecules were again docked using the tools Hex server, iGEMDOCK, FireDock and SwissDock. The docked results were statistically analysed following DST and Zhang rule and selected the top ranked molecules from each plant viz. 2-methyl-6-(4-hydroxy-3-methylphenyl)-2-hepten-4-one from C. longa, Alpha-ylangene from E. cardamomum and Farnesal from Z. officinale as lead molecules. Mature seeds of E. cardamomum and mature rhizomes of Z. officianale and C. longa were air dried and extracted with 99% ethanol using a Soxhlet apparatus for 6-8 hours. The extracts were concentrated to dryness using a rotary evaporator and tested anti-mycobacterial activity by Luciferase reporter phage (LRP) assay against standard strain of M. tuberculosis H37RV at three different concentrations (25, 250 and 500 μg/ml). The results revealed that all the three plants have potential antituberculosis activity. In the order of merit Z. officinale rank first E. cardamomum rank second and C. longa rank third respectively. The results revealed the efficacy of anti-tuberculosis activity and the responsible phytomolecules in each plant. It also insights the discovery of novel drugs with desirable qualities from these plants that should be safe, effective and affordable to the poor people.