In trangenic rice, α- and β-tubulin regulatory sequences control GUS amount and distribution through intron mediated enhancement and intron dependent spatial expression

Abstract

The genomic upstream sequence of the rice tubulin gene OsTub6 has been cloned, sequenced and characterized. The 5'UTR sequence is interrupted by a 446 bp long leader intron. This feature is shared with two other rice beta-tubulin genes (OsTub4 and OsTub1) that, together with OsTub6, group in the same clade in the evolutionary phylogenetic tree of plant beta-tubulins. Similarly to OsTub4, the leader intron of OsTub6 is capable of sustaining intron mediated enhancement (IME) of gene expression, in transient expression assays. A general picture is drawn for three rice alpha-tubulin and two rice beta-tubulin genes in which the first intron of the coding sequence for the formers and the intron present in the 5'UTR for the latters, are important elements for controlling gene expression. We used OsTua2:GUS, OsTua3:GUS, OsTub4:GUS and OsTub6:GUS chimeric constructs to investigate the in vivo pattern of beta-glucuronidase (GUS) expression in transgenic rice plants. The influence of the regulatory introns on expression patterns was evaluated for two of them, OsTua2 and OsTub4. We have thus characterized distinct patterns of expression attributable to each tubulin isotype and we have shown that the presence of the regulatory intron can greatly influence both the amount and the actual site of expression. We propose the term Intron Dependent Spatial Expression (IDSE) to highlight this latter effect.

The 5′-UTR intron of the midgut-specific BmAPN4 gene affects the level and location of expression in transgenic silkworms

“…Although IDSE has been reported in some plant studies, the mechanism is still not clear (Giani et al, 2009;Jeong et al, 2007Jeong et al, , 2006Lu et al, 2008). The first intron of PhADF1 changes tissue-specific expression and deleting the 5 0 -upstream region alters the expression pattern (Jeong et al, 2007).…”

“…The 5UIs are important regulatory elements for controlling gene expression and are reported to change expression levels and locations in plants (Bicknell et al, 2012;Chorev and Carmel, 2012;Giani Our previous study showed that BmAPN4 is expressed in the entire midgut but expression of EGFP under the promoter P3P is only in the anterior midgut of P3 silkworms (Lu et al, 2012). To determine if the difference in expression was due to the 5UI of BmAPN4, we cloned P3Pþ5UI, which consisted of P3P and 5UI, and generated transgenic P3þ5UI silkworms (Figs.…”

“…Many 5UIs enhance gene expression (Aguilar-Hernandez and Guzman, 2013;De La Torre and Finer, 2015;Rose, 2008) in an effect termed intron-mediated enhancement (IME) (Mascarenhas et al, 1990). Some 5UIs influence the spatial pattern of gene expression (Giani et al, 2009;Lu et al, 2008) in a mechanism termed intron-dependent spatial expression (IDSE) (Giani et al, 2009). Several studies have reported on IME and IDSE in plants (Bicknell et al, 2012;Chorev and Carmel, 2012;Jeong et al, 2007Jeong et al, , 2006Le Hir et al, 2003); however, few studies have been reported in insects.…”

Strategies to Increase Heterologous Protein Expression in Rice Grains

“…The upstream 5′UTR intron of α-tubulin genes is responsible for their expression (Fiume et al 2004). The 5′UTR intron in rice β-tubulin gene Ostub16 has also been used to enhance expression levels in rice (Giani et al 2009). Both transient expression (Hwang et al 2002) and transgenic analyses in rice seeds have showed a high level of target protein expression with the use of a portion of the native 5′UTR sequence of Gt1.…”

Differences in intron-mediated enhancement of gene expression by the first intron of cytosolic superoxide dismutase gene from rice in monocot and dicot plants

“…It is shown that introns also participate in the control of tissue speci city (Fu et al 1995;Gianì et al 2009;Jeon et al 2001;Jeong et al 2006) as well as the control of the expression level.…”