Development of Gene Constructs for Studying Expression Pattern of TA29 Promoter and coxIV Presequence
KrishiKosh
View Archive InfoField | Value | |
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
|
|