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Development of Hairpin Construct for Silencing of 12 Desaturase Gene in Cotton Seed Oil

KrishiKosh

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Title Development of Hairpin Construct for Silencing of 12 Desaturase Gene in Cotton Seed Oil
 
Creator Sanjay Kumar Bhariya
 
Contributor H.M. Vamadevaiah
 
Subject Plant Biotechnology
 
Description Cotton (Gossypium spp) is most important crop for fibre production but still cotton is
the fifth largest crop in terms of edible oilseed production in the world. In addition to 21%
oil, cottonseed is a source of relatively high-quality protein (23%). However, the ability to
use this nutrient-rich resource for food is hampered by the presence of high palmatic
and linoleic acid forms saturated fatty acid and later is thermostablity polyunsaturated fatty
acid. Post transcriptional silencing of 12 desaturase gene was envisaged as a way to activate
silencing mechanism in cotton, thereby blocking the formation of linoleic acid production
and accumulation of oleic acid in the transformants. Hairpin RNA mediated technique is a
method of PTGS is a specific RNA mechanism in plants that takes care of aberrant unwanted
excess of its own or foreign RNA intracellularly in a homology dependent manner.
In this study, an attempt was made to develop construct for silencing of 12
desaturase gene, and analyse its expression in tobacco plants. A 600 bp DNA fragment was
amplified using 12 desaturase gene specific primer from cotton ovules after 30 days
pollination. The amplicon was cloned in pTZ57R/T and transformed into E. coli DH5 .
Transformants were confirmed through PCR and restriction. The analysis of the sequence
revealed maximum of 100% homology at nucleotide and 100% homology at amino acid level
with reported sequence in the database (AF270425). Partial 12 desaturase gene was cloned
in both sense and antisense direction in generic ihp vector. The expression cassette carrying
insert from generic ihp vector was then subcloned into plant transformation vector
pCAMBIA 1305.1 to facilitate plant transformation. The recombinant clones were then
mobilized into Agrobacterium tumefaciens LBA4404 by tri parental mating then transformed
into tobacco plants. Callus was analyzed through histochemical analysis of gus gene
exression against control plants.
 
Date 2016-11-21T18:58:44Z
2016-11-21T18:58:44Z
2012
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/86841
 
Format application/pdf
 
Publisher UAS, Dharwad