1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)
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Item Cloning and characterization of coat protein gene of Tomato leaf curl virus infecting tomato and its phylogenetic relationship with other members of geminiviridae(Department of Plant Biotechnology, College of Agriculture , Vellayani, 2022-10-27) Athira ,S M; Ayisha ,RThe study entitled ‘Cloning and characterization of coat protein gene of Tomato leaf curl virus infecting tomato and its phylogenetic relationship with other members of Geminiviridae’ was carried out at College of Agriculture, Vellayani during the year 2021- 2022. The objective of the study was molecular characterization and cloning of coat protein gene of Tomato leaf curl virus (ToLCV) infecting tomato and its phylogenetic analysis with other members of Geminiviridae. Symptomatology of virus infected tomato plants was studied. Infected plants were collected from different regions of Vellayani campus, Kerala Agricultural University and the virus was maintained by graft inoculation. The virus was serologically characterized using DAS-ELISA (Double Antibody Sandwich-ELISA) and DIBA (Dot Immuno Binding Assay) using ToLCNDV (Tomato leaf curl New Delhi virus) antisera and higher viral titer was shown by plants with severe reduction in leaf size (8-fold absorbance value). Genomic DNA was extracted from the infected samples, and coat protein (CP) gene-specific primers were used for molecular detection. PCR yielded expected amplicon size of 500bp and 600bp and were sequenced. The BLAST analysis of the sequence revealed similarities with Tomato leaf curl Palampur Virus (ToLCPMV) and ToLCNDV of 95% and 94%, respectively. Both bipartite virus and monopartite virus with a satellite DNA were detected by rolling circle amplification (RCA), which was followed by Restriction Fragment Length Polymorphism (RFLP). PCR was done using RCA fragments as template with DNA A specific primers and the amplicons obtained were cloned. Sequencing of cloned genes followed by BLAST analysis showed 98.61 per cent similarities with ToLCPMV isolates. Phylogenetic analysis of partial DNA A gene of Vellayani isolate with other strains of ToLCV showed close relation to ToLCPMV infecting cucurbits. Comparitive analysis of partial DNA A sequence with other viruses in genera Begomovirus showed closest relation with Melon leaf curl virus and Cotton leaf curl virus from Pakistan. Comparison of partial amino acid sequence of CP of ToLCV Vellayani isolates with other mono and bipartite begomoviruses revealed a maximum of 99.11 per cent similarity with pre coat protein genes of ToLCPMV. According to the current investigation, the Begomovirus that infects tomatoes in the Vellayani region is bipartite as well as monopartite with a satellite DNA. The CP and DNA A genome phylogenetic analyses revealed a strong relationship between the Vellayani isolate and the ToLCPMV isolates infecting cucurbitsItem Isolation, cloning and molecular characterization of defensin gene(s) in black pepper (Piper nigrum L.)(Department of Molecular Biology and Biotechnology, College of Agriculture ,Vellayani, 2023-03-22) Abirami, G; Anuradha, TPlant defensins are cationic antimicrobial peptides that function in the innate immune response in plants. Many plant defensins having antimicrobial activity are molecularly characterized in different plants. However, defensin genes present in black pepper are not completely characterized. The study entitled “Isolation, cloning and molecular characterization of defensin gene(s) in black pepper (Piper nigrum L.)” was conducted at the Department of Molecular Biology and Biotechnology, College of Agriculture Vellayani, Thiruvananthapuram during 2021-2022. The objective of this study was isolation, cloning and molecular characterization of antimicrobial peptide defensin gene(s) from black pepper variety Panniyur 1. In this study, the full-length gene sequence of a novel defensin gene termed PnDef was isolated for the first time from black pepper var. Panniyur 1 using gene specific primers. The full length of PnDef gene is found to be 356bp. The coding sequences of PnDef was deduced using Genscan software, which was found to contain 231bp open reading frame encoding a protein of 76 amino acid residues. The blast analysis of PnDef amino acid sequence showed 72.60% similarity with the defensin protein of Pyrus ussuriensis × Pyrus communis. The phylogenetic analysis showed a close relationship of PnDef to defensin protein from Rosaceae family. The clustal analysis of PnDef revealed eight conserved cysteine residues in similar positions. The putative PnDef mature protein showed striking similarity with other plant defensins representing a small molecular weight of 5.213 kDa, theoretical isoelectric point (pI) of 8.95 with an aliphatic index and GRAVY of 60.26 and 0.154 respectively. It has a potential 29 residue signal peptide and the remaining 47 residues belong to the mature peptide region. The secondary structure analysis of PnDef revealed that it has 46% alpha helix and 25% of β-sheet structures. The three-dimensional structure modeling of PnDef showed a striking resemblance (81%) with the antifungal defensin of Medicago trancatula implying it may have similar antimicrobial activity. PnDef was found to have three conserved disulfide bonds formed between six cysteine residues. In silico analysis of the PnDef indicated antibacterial, antiviral and antifungal properties of the peptide.Using the sequence data identified in this study one can further go for expression profiling of black pepper defensin in different plant parts under biotic and abiotic stress conditions, recombinant protein can be isolated by inserting into the expression vector and using the recombinant protein one can identify the inhibition activity of the protein against plant pathogens under in vitro studies. Molecular docking can also be done prior to the in vitro studies using the 3D structure of black pepper defensin to identify potential pathogen targets.