Development of an organism is strictly regulated by molecular and genetic mechanisms. The precise regulatory processes involving switching on or off of certain set of genes at a specific stage and place mediated by certain combination's of regulatory elements are fundamental to the development. Identification and isolation of plant genes and promoters are essential in plant research communities to understand the biological processes at molecular level. Identification and isolation are also beneficial in studying genetic expression in transgenic plants. So it is important to have a deep knowledge and understanding of regulatory mechanisms at gene level to select the best promoter for use in transgenic plants.
A promoter is a regulatory element that drives the expression of introduced genes in transgenic plants that are of agricultural and economic importance in a specific manner. So there is a need to identify and isolate promoters that regulates tissue specific and stage specific expression of genes in plants. Trichomes are specialized epidermal cells that provide first line of defense against insects and pests. In order to develop transgenic plants that are resistant to pests and insects attack .To isolate trichome specific promoter T-DNA insertional mutagenesis has been used. In this approach a binary vector (pGKB5) containing promoter less reporter gene B-glucuronidase (uidA) adjacent to right border of T-DNA is used to create mutant Arabidopsis thaliana plants.
Arabidopsis thaliana is an angiosperm, dicot plant of Brassicaceae family, which is also a model organism in plant kingdom. It is useful for making transgenic plants because of its small genome, small size, easy growth in lab in a relatively small space, development is also rapid (only 5-6 weeks from seed germination to production of a new crop of seeds) and very little repetitive DNA. On screening of mutant plants there is an expression of GUS gene exclusively in trichomes. Due to t-DNA insertional mutagenesis, sequences upstream to GUS gene acts as a promoter, which drives its expression in trichomes.
[...] Jefferson, R.A, Kavanagh, T.A and Bevan, M.W GUS fusions: β- glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 3901–3907. Jeon, J.S., Lee, S., Jung, K.H., Jun, S.H., Jeong, D.H., Lee, J., Kim T-DNA insertional mutagenesis for functional genomics in rice. Plant J. 22: 561-570. Kim, Y. and An, G Pollen-specific expression of the Arabidopsis thaliana α1-tubulin promoter assayed by β-glucuronidase, chloramphenicol acetyltransferase and diphtheria toxin reporter genes. Transgenic Res. 18 Different temporal and spatial gene expression patterns occur during anther development. [...]
[...] If GUS assay results in the expression of GUS gene specifically in trichomes then the sequence that was identified through T-DNA insertional mutagenesis will be confirmed as promoter that is responsible for expression of GUS gene in trichomes. Future prospects in this area are very bright. Since trichomes acts as first line of defense against insect and pests attack, it is possible through cloning to create transgenic plants with such a stringent defense system that could not be broken by insects & pests. [...]
[...] All the primers designed for synthesis were gene specific primers and the lengths varied between 20-30 nucleotides. The primer sequences were checked against the Arabidopsis genome sequence by using the BLAST program so as to avoid non-specific primer binding during amplification. In most of the primers, suitable restriction enzyme sites were attached at the end of the primers so as to facilitate directional cloning into the binary plant transformation vector. All the primers were synthesized from the GCC Brotech and were dissolved in freshly prepared and sterilized 10 mM Tris-Cl (pH 7.0 ) to a final concentration of 100 μM stocks. [...]
[...] Tyagi, A.K Plant genes and their expression. Curr. Sci. 80: 161-169. Van Haaren MJ & Houck CM A functional map of fruit specific promoter of the tomato 2A11gene. Plant Mol Biol. 21: 625-640. Wei, W., Twell, D. and Lindsey, K A novel nucleic acid helicase gene identified by promoter trapping in Arabidopsis. Plant J. 11: 1307-1314. Yanofsky, M.F., Ma, H., Bowman, J.L., Drews, G.N., Feldman, K.A. and Meyerowitz, E.M The protein encoded by the Arabidopsis homeotic gene Agamous resembles transcription factors. Nature [...]
[...] The promoter tagged through T-DNA insertional mutagenesis could be isolated using PCR based techniques like Thermal Asymmetric Interlaced PCR (TAIL-Liu et al., 1995), Inverse PCR (IPCR-Ochman et al., 1988) and plasmid rescue (Yanofsky et al., 1990) form mutant plant exhibiting GUS gene expression in tissue of interest. Several promoters have been isolated using promoter trap vectors in Arabidopsis. Some of them are At Em promoter (embryo specific expression, Wei et al., 1997), HVT1 promoter (tapetum & vascular specific expression, Wei et al., 1997), Pyk 20 promoter (nematode feeding structure expression, Puzio et al., 2000), cryptic promoter (guard cell specific expression, Mollier et al., 2000), functionally redundant development related genes (Prasad et al., 2005), cryptic root specific promoter etc. [...]
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