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Development of Gene Constructs for Studying Expression Pattern of TA29 Promoter and coxIV Presequence

KrishiKosh

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Title Development of Gene Constructs for Studying Expression Pattern of TA29 Promoter and coxIV Presequence
 
Creator KALYANI KALLAM
 
Contributor Anuradha, G
 
Subject Development, Gene, Constructs, Studying, Expression, Pattern, TA29, Promoter, coxIV, Presequence
 
Description The present work was carried out with the basic idea of developing gene
constructs for studying expression pattern and tissue specificity of tapetum specific
promoter TA29 from tobacco and mitochondrial targeting efficiency of yeast cytochrome
C oxidase subunit (COXIV) in safflower with the use of reporter gusA gene (β-
glucuronidase) from E.coli. At Directorate of Oilseeds Research, Hyderabad, constructs
for inducing male sterility in safflower by tapetum specific expression of ORFH522 in
mitochondria using coxIV presequence have been developed. As a preliminary work, it
was intended to study expression of TA29 promoter and efficiency of coxIV presequence
in safflower with the use of β-glucuronidase reporter system.
With the above strategy, the present work progressed in four phases, which
included molecular as well as tissue culture studies. In the first phase, development of
appropriate gene constructs was taken up. Three constructs were developed in the generic
vector background pCAMBIA1391Z, which had gus and polyA. The constructs include
CaMV35S-coxIV cloned in pCAMBIA1391Z making CaMV35S-coxIV-gus-polyA
cassette and a control vector for this with CaMV35S alone without coxIV in
pCAMBIA1391Z obtaining CaMV35S-gus-polyA construct. The third construct includes
cloning of TA29 promoter in front of gus gene to obtain the cassette TA29-gus-polyA.
All the constructs were confirmed through colony PCR and restriction analysis.
The second phase included mobilization of all the three constructs along with
pCAMBIA1391Z as control into Agrobacterium strain for subsequent transfer into
safflower. The constructs in Agrobacterium were further confirmed through PCR and
restriction analysis.
The third phase of this work involved transformation of safflower with the
confirmed Agrobacterium clones (containing the recombinant binary vectors) and
development of the transgenics. The final phase of this work involved molecular
confirmation of the developed transgenic plants using PCR based techniques.
The future line of work with these transgenics involves studies on mitochondrial
targeting efficiency of coxIV and tapetal tissue specificity of TA29 promoter in
safflower, using gus gene expression as the marker by using appropriate techniques.
Results with these studies will give a precise view of the effectiveness of coxIV and
TA29 promoter, which will further strengthen the utility of orfH522 based constructs for
induction of male sterility in safflower.
 
Date 2016-08-19T13:39:45Z
2016-08-19T13:39:45Z
2005
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/73020
 
Language en
 
Relation D7779;
 
Format application/pdf
 
Publisher ACHARYA N.G. RANGA AGRICULTURAL UNIVERSITY, RAJENDRANAGAR, HYDERABAD