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Designing pathogen inducible synthetic promoters and functional validation of a new Eukaryotic promoter - probe vector

KrishiKosh

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Title Designing pathogen inducible synthetic promoters and functional validation of a new Eukaryotic promoter - probe vector
 
Creator G.M.Raveendra
 
Contributor Sumangala.Bhat
 
Subject Plant Biotechnology
 
Description In this study, an attempt was made to design pathogen inducible promoters, and
construct and functionally validate a new promoter-probe vector. W 2 X, GCC 2 X, GCC 3
X, S 2 X, Myb 2 X pathogen inducible promoters contained various pathogen-responsible
cis-regulatory elements in multiple copies fused to a minimal promoter (-46 region of CaMv
35S promoter) at 3’, with a spacer sequences in between. They were synthesized at
GENEART, Germany.
A promoter-probe vector (pRR21) was constructed in binary vector (pCAMBIA
1305.1) background with an improved version (SgfpS65T) of GFP and Nos-ter. pRR21
carried a multiple cloning site with target sequences for BamHI, XbaI, SalI, PstI and HindIII
to clone any DNA fragment whose promoter activity is to be checked. Complete sequence
and feature (annotation) information of pRR21 has been deposited at GenBank of NCBI with
an accession number (EU760495). This promoter-probe vector constructed with SgfpS65T.
because pRR21 is in a binary vector background, it is expected to work in many plant
systems.
Promoter-probe vector was functionally validated by PCR cloning CaMV 35S
promoter from pWBVec8 into the multiple cloning site of pRR21 to get pRR20. Similarly, all
five synthetic pathogen inducible promoters were cloned into pRR21. The recombinant
promoter-probe vectors were transferred into Agrobacterium by tri-parental mating. Of the 30
tobacco leaf discs co-cultivated with Agrobacterium carrying pRR20, 19 showed SgfpS65T
expression when observed under the fluorescent microscope at wavelength of 480-520 nm.
De novo calli formed from all SgfpS65T positive leaf discs also showed fluorescence.
Functional validation and expression analysis of pathogen inducible synthetic promoter is to
be carried out.
 
Date 2016-10-26T16:52:27Z
2016-10-26T16:52:27Z
2008
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/82326
 
Format application/pdf
 
Publisher UAS, Dharwad