In a genome sequence, promoter designates to an upstream region of transcription start site (TSS) of a gene (de Boer et al., 1999; Ilham et al., 2003), although downstream sequence may also affect transcription initiation. Several functional elements have been identified as promoter constituents for precise and regulated transcriptional initiation: TATA box, CCAAT box, cap signals, GC-box, Initiator (Inr) motif, Downstream Promoter Element (DPE, from drosophila), TFIIB-Recognition Element (BRE), and so-called cis-regulatory elements. Promoters of several housekeeping genes lacks TATA box and expression is initiated through "inr" elements (Smale, 2001).
Regulation of gene expression involves a complex molecular network. Transcription of genes is controlled primarily by TFs identifying and binding to speciﬁc short DNA sequence motifs in the cis-regulatory region (promoter and enhancer) of these genes, leading to activation or repression of transcription in response to changes in the environment as well as during development. Based on the nature of gene expression, promoters have been broadly classified as constitutive, tissue specific, temporal/stage specific or inducible promoters (Gurr and Rushton, 2005). One of the applied aspects of plant promoter studies have led to designing of synthetic promoters, basically through rearrangement of cis-acting sequences, for targeted engineering of transcriptional regulation of genes (Venter, 2007).
Strategies for plant promoter isolation
One of the major bottlenecks in conventional approach of promoter isolation is due to cumbersome process of specific identification of promoter within large gene families. Even the recently developed techniques like microarray and real time PCR-based approach of transcript identification is comparatively technically demanding and also limited with insensitivity to detect transiently or less expressing genes (Springer 2000). In this context a complementary approach of plant promoter isolation take advantage of random insertion of reporter genes either through T-DNA or transposons as mutagen (Datla et al. 2008, Gupta et al., 2012). In principle the reporter gene gets activated only when inserted under the influence of a regulatory sequence. The reporter gene in a T-DNA or transposon in the process literally traps or tags an element conferring the expression behavior. Difference in the 'trapping' constructs predominantly dictates the type of DNA element identified whether genes, exons, intron, or the regulatory elements like promoters and enhancers. In promoter trapping approach a promoterless reporter gene along with a selectable marker is largely used to clone a desired promoter sequence from the mutant lines screened under specific condition.
Several techniques are available to isolate and clone the T-DNA flanking genomic DNA sequences from the mutant population. Employing one of the following methods will allows cloning of the upstream sequence of the T-DNA insertion site consisting of the putative promoter elements.
i) Screening of the genomic DNA library constructed from the mutant plant
In this approach the DNA fragments flanking the T-DNA are identified from the library and used as a probe to isolate the wild type genomic sequence (Marks and Feldman 1989).
ii) Plasmid rescue
It is an approach which is employed in case the T-DNA construct consists of an antibiotic resistance gene and a bacterial origin of replication (ori). The genomic DNA of mutant plant is subjected to complete digestion followed by ligation to circularize all the fragments and transform them into E. coli host. The plasmids isolated from the E. coli would be analyzed for the presence of T-DNA and the flanking plant DNA sequences (Yanofsky et al. 1990).
iii) Inverse PCR (IPCR)
The IPCR strategy principally involves cleavage of genomic DNA by a suitable enzyme followed by ligation of the fragments to facilitate self-circularization. A set of nested primers derived from the T-DNA border regions are used to amplify the flanking DNA, cloned and sequenced (Ochman et al. 1988).
iv) The thermal asymmetric interlaced PCR (TAIL-PCR)
The technique makes use of three nested T-DNA-specific primers in one end and a short arbitrary degenerate (AD) primer in the other end. Three different PCR reactions are performed with these primer sets. The primary PCR reaction involves different primer annealing temperatures and low and high stringent cycles to facilitate annealing of arbitrary and specific primers, respectively. This step results into both specific as well as nonspecific amplification of products. In the next two steps of PCR reactions the non-specific products are eliminated amplifying predominantly the T-DNA flanking genomic DNA (Liu et al. 1995).
(v) Genome Walking
The other PCR-based method of promoter cloning includes enzymatic blunting of mutant genomic DNA and ligation of adapters followed by amplification of the T-DNA flanking DNA using two nested primers specific to T-DNA and adapters (Siebert et al. 1995).
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