POST-TRANSCRIPTIONAL GENE SILENCING (PTGS) OF TOMATO LEAF CURL VIRUS (ToLCV) IN TOMATO
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Date
2007-08-30
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University of Agricultural Sciences GKVK, Banglore
Abstract
Tomato, an economically important crop in many countries is
plagued by many viral diseases including leaf curl caused by Tomato leaf
curl virus (ToLCV) belonging to the genus begomovirus. Begomoviruses
are small circular, single stranded DNA plant viruses. Yield losses usually
vary from 28-92% making tomato cultivation unprofitable. Genetically
engineering resistance is a viable alternative is to genetically engineer
tomato for protection against ToLCV. PTGS/RNAi is a novel gene
regulatory mechanism that limits the transcript level by either
suppressing transcription or by activating a sequence-specific RNA
degradation process.
We have cloned and characterized ToLCV-coat protein (TCP),
replicase (TRP) and suppressor of PTGS (TRS) genes from a Dharwad local
isolate. Constructs have been developed using all the three genes for the
available gene silencing strategies viz., sense (s), antisense (as), ihp (sas)
and HUTR (heterologous 3'-untranslated region). Coat protein (TCP) gene
has been cloned and expressed in a prokaryotic system. As a comparative
study plant expression vectors carrying TCP, TRP and TRS gene following
different strategies were used for transgenic development through
Agrobacterium. Analysis of putative To-transgenics showed positive for
PCR, GUS. Dot blot and Southern blot analysis. Semi-quantitative PGR
analysis of plants from TRP constructs showed drastic reduction in the
virus inoculum compared to non-transgenic plants. The Ti-generataion
transgenic plants obtained from TRP constructs were positive for PCR and
Dot blot analysis. Among different strategies tested for resistance to
ToLCV in transgenics, those with sas/ihp construct showed significant
resistance against ToLCV followed by HUTR, antisense (as) and sense (s).
Similarly, among the three different genes tested, silencing was more in
TRS constructs followed by TRP and TCP.
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