Athira S M
Cloning and characterization of coat protein gene of Tomato leaf curl virus infecting tomato and its phylogenetic relationship with other members of geminiviridae - Vellayani Department of Plant Biotechnology, College of Agriculture 2022 - 74p.
BSc - MSc(Int.)
The 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 cucurbits.
Plant Biotechnology
660.6 / ATH/CL PG
Cloning and characterization of coat protein gene of Tomato leaf curl virus infecting tomato and its phylogenetic relationship with other members of geminiviridae - Vellayani Department of Plant Biotechnology, College of Agriculture 2022 - 74p.
BSc - MSc(Int.)
The 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 cucurbits.
Plant Biotechnology
660.6 / ATH/CL PG