Abstract
Promoter trapping is a method used to isolate and characterize regulatory regions from genomes by elucidating the expression of a promoterless reporter gene flanked by two transposable elements. The conventional method of promoter trapping requires the generation of stable transformed tissue culture derived plant lines. Nevertheless,this method can be laborious, time consuming and expensive. As an alternative method, leaf disc samples produced via leaf agroinfiltration was employed in this work. A promoter trapping construct named pCAMDIN was created which contains a promoterless GUS (?-glucuronidase) gene flanked by the left and right T-DNA border using pCAMBIA 1301 and pBI 121. Following that,the protocol for agroinfiltration of tomato plants using both direct agroinfiltration and vacuum agroinfiltration was optimized. Non-destructive protocols for detection of GUS genes were tested and optimised. Following that, GUS gene expression was studied and areas which showed expression were isolated by making leaf punches. Analysis was carried out by Southern blotting and T-DNA fingerprinting to determine the gene copy number.
Highlights
Promoters control the fundamental pattern of gene expression in an organism and are of great interest for understanding plant gene expression patterns leading to wide interest in their uses (Yang et al, 2004)
In order to study the functional genomics of plants, insertional mutagenesis is used as a powerful tool which allows for genome-wide identification and isolation of functionally redundant genes
Insertional mutagenesis to isolate and characterize plant regulatory sequences can be done via ethylmethanesulfonate (EMS), fastneutron treatment and transposable elements such as T-DNA (Walden, 2002)
Summary
Promoters control the fundamental pattern of gene expression in an organism and are of great interest for understanding plant gene expression patterns leading to wide interest in their uses (Yang et al, 2004). Promoter trapping is a method used to detect the presence of promoter sequences within genomic regions where T-DNA is used to insert mobile elements containing a promoterless reporter gene flanked by transposable elements into the plant genome (Sundaresan et al, 1995). Analysis of GUS expression will enable the detection, isolation and sequencing of the promoter region from the upstream location of the T-DNA integration.
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