Strategies for Development of Functionally Equivalent Promoters with Minimum Sequence Homology for Transgene Expression in Plants: cis-Elements in a Novel DNA Context versus Domain Swapping

Abstract

The cauliflower mosaic virus 35S (35S) promoter has been extensively used for the constitutive expression of transgenes in dicotyledonous plants. The repetitive use of the same promoter is known to induce transgene inactivation due to promoter homology. As a way to circumvent this problem, we tested two different strategies for the development of synthetic promoters that are functionally equivalent but have a minimum sequence homology. Such promoters can be generated by (a) introducing known cis-elements in a novel or synthetic stretch of DNA or (b) "domain swapping," wherein domains of one promoter can be replaced with functionally equivalent domains from other heterologous promoters. We evaluated the two strategies for promoter modifications using domain A (consisting of minimal promoter and subdomain A1) of the 35S promoter as a model. A set of modified 35S promoters were developed whose strength was compared with the 35S promoter per se using-glucuronidase as the reporter gene. Analysis of the expression of the reporter gene in transient assay system showed that domain swapping led to a significant fall in promoter activity. In contrast, promoters developed by placing cis-elements in a novel DNA context showed levels of expression comparable with that of the 35S. Two promoter constructs Mod2A1T and Mod3A1T were then designed by placing the core sequences of minimal promoter and subdomain A1 in divergent DNA sequences. Transgenics developed in tobacco (Nicotiana tabacum) with the two constructs and with 35S as control were used to assess the promoter activity in different tissues of primary transformants. Mod2A1T and Mod3A1T were found to be active in all of the tissues tested, at levels comparable with that of 35S. Further, the expression of the Mod2A1T promoter in the seedlings of the T 1 generation was also similar to that of the 35S promoter. The present strategy opens up the possibility of creating a set of synthetic promoters with minimum sequence homology and with expression levels comparable with the wild-type prototype by modifying sequences present between cis-elements for transgene expression in plants.

Cis-regulatory elements used to control gene expression in plants

“…Multiplication of the elements, their reorganization and ligation with synthetic and native cis sequences are used to obtain synthetic promoters (Bhullar et al 2003;Mehrotra et al 2011;Venter and Botha 2010). An optimal plant synthetic promoter should have precisely defined specificity, immediate inducibility, the versatility of applications and should guarantee the best efficiency of transformation (Venter and Botha 2010).…”

Functional Analysis of Synthetic Promoters Containing Pathogen-Responsive <i>cis</i>-Elements

“…Homology-dependent gene silencing (HDGS) is of great concern in plant genetic engineering strategies and is thought to be caused by multiple copies of homologous transgene and promoter sequences (Matzke et al, 2002). Repetitive use (three copies) of cis-elements with identical core-sequences and homologous intervening regions (within a functional domain) might also cause depletion of transcription factors, consequently reducing endogenous gene expression (Bhullar et al, 2003). Therefore, design strategies using multimers of cis-motifs need to be optimized to achieve the desirable inducibility of the transgene.…”

Subsídios à transformação genética de plantas de Catasetum pileatum (Orchidaceae) por meio de tecidos merismáticos radiculares e caulinares

“…Promotores podem também ser construídos artificialmente, contendo regiões regulatórias apropriadas e seqüências ativadoras (Bhullar et al, 2003, Potenza et al, 2004.…”

“…Inicialmente descrito por Odell et al, em 1985, este promotor vem sendo utilizado intensamente em pesquisas de transformação genética, devido a este proporcionar expressão intensa e constitutiva em praticamente todos os tecidos e órgãos de muitas espécies, tanto dicotiledôneas quanto monocotiledôneas (Bhullar et al, 2003). Além deste fato, este promotor é amplamente disponível comercialmente, podendo ser encontrado em diversas construções vetoriais para transformação genética (Potenza et al, 2004).…”

“…A despeito das vantagens já citadas, encontra-se na literatura a menção de certos problemas causados pela utilização deste promotor, como é o caso do silenciamento de transgenes por ele regulados em muitos casos de transformações genéticas estáveis (Elmayan & Vaucheret, 1996;Angell & Baulcombe, 1997;Mishiba et al, 2005a;Bhullar et al, 2003). Mishiba et al (2005a) (Matzke, Mette & Matzke, 2000).…”