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CHARACTERIZATION OF CITRUS TRISTEZA VIRUS ISOLATES ORIGINATING FROM UTTARAKHAND AND EXPRESSION OF THE COAT PROTEIN GENE

KrishiKosh

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Title CHARACTERIZATION OF CITRUS TRISTEZA VIRUS ISOLATES ORIGINATING FROM UTTARAKHAND AND EXPRESSION OF THE COAT PROTEIN GENE
 
Creator JAYWANT KUMAR SINGH
 
Contributor Kajal Kumar Biswas
 
Subject You have exceeded your usage quota. Please contact gshukla [at] iitk.ac.in
 
Description T-8459
Citrus tristeza virus (CTV) belonging to the genus Closterovirus, predominantly transmitted
by aphid vector, Toxoptera citricida is one of the most destructive pathogen in most of the
citrus growing countries in the world including India. CTV genome is a single stranded
positive sense genomic RNA (gRNA) of 19.3 kb length with 12 open reading frames (ORFs)
encoding at least 19 putative proteins. To detect and characterize CTV, survey was conducted
in two orchards of acid lime cv. Pant lemon in experimental farm of GBPUA&T, Pantnagar,
Uttrakhand. ELISA test detected Pant lemon orchards to be CTV infected. Percent tree
infection was estimated which ranges from 16.6-20.5%. RT-PCR using universal primer
pairs, KK488(F) and KK491(R) confirmed CTV infection in the plants. One CTV isolate,
Pant 4 was biologically indexed to determine its potential host range using eight available
citrus hosts; Kinnow, Nagpur and Darjeeling mandarin, Kagzi lime, Rough lemon, sweet
lime, sour orange and sweet orange. Isolate Pant 4 infected all the citrus hosts, except Rough
lemon, but all the mandarin hosts remained asymptomatic showing only poor growth. Two
Pantnagar isolates, Pant 4 and Pant 5 were considered for further molecular study using five
pairs of overlapping primers amplifying 3038 nt fragment of 5′ORF1a and four pairs
amplifying of ORF7 to ORF10 of CTV genome. The amplified sequences of Pantnagar CTV
genomes were cloned, sequenced and sequences were analysed and compared with other
Indian and international isolates. Sequence comparison and phylogenetic analysis in present
study shows that Indian isolate Pant 4 is similar to previously reported Indian isolate Kpg3
based on 5'ORF1a, 3038 nt (98% identity), ORF7 (98% identity), ORF9 (99% identity) and
ORF10 (100% identity), but different from another isolate B165 reported from South India.
In analysis of 404 nt 5'ORF1a fragment, Pant 4 and Pant 5 were similar but for ORF 8, Pant
5 was distant from Kpg3, thus showing that isolate Pant 4 is different from Pant 5; indicating
genetic diversity in CTV isolates of Pantnagar region. Recombination detecting program
RDP3 using assembled 4619 nt sequence (5′ORF1a 3038 nt fragment plus a total 1581 nt of
ORF7, ORF9 and ORF10) showed isolate Pant 4 to be recombinant, involving Israel isolate
VT as major and Hawaii isolate HA16-5 as minor parent. A complete CP gene of CTV
isolate Pant 4 was sub-cloned in expression vector pET-28a (+) using site directed primer set,
KK547(F) and KK548(R). The recombinant coat protein (rCP) gene was used for protein
expression in E. coli strain BL21 cells.
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Date 2016-11-22T11:57:50Z
2016-11-22T11:57:50Z
2011
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/86899
 
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