Unprecedented enhancement of transient gene expression from minimal cassettes using a double terminator

Abstract

The potential of using vector-free minimal gene cassettes (MGCs) with a double terminator for the enhancement and stabilization of transgene expression was tested in sugarcane biolistic transformation. The MGC system used consisted of the enhanced yellow fluorescent protein (EYFP) reporter gene driven by the maize ubiquitin-1 (Ubi) promoter and a single or double terminator from nopaline synthase (Tnos) or/and Cauliflower mosaic virus 35S (35ST). Transient EYFP expression from Tnos or 35ST single terminator MGC was very low and unstable, typically peaking early (8-16 h) and diminishing rapidly (48-72 h) after bombardment. Addition of a ~260 bp vector sequence (VS) to the single MGC downstream of Tnos (Tnos + VS) or 35ST (35ST + VS) enhanced EYFP expression by 1.25- to 25-fold. However, a much more significant increase in EYFP expression was achieved when the VS in 35ST + VS was replaced by Tnos to generate a 35ST-Tnos double terminator MGC, reaching its maximum at 24 h post-bombardment. The enhanced EYFP expression from the double terminator MGC was maintained for a long period of time (168 h), resulting in an overall increase of 5- to 65-fold and 10- to 160-fold as compared to the 35ST and Tnos single terminator MGCs, respectively. The efficiency of the double terminator MGC in enhancing EYFP expression was also demonstrated in sorghum and tobacco, suggesting that the underlying mechanism is highly conserved among monocots and dicots. Our results also suggest the involvement of posttranscriptional gene silencing in the reduced and unstable transgene expression from single terminator MGCs in plants.

A novel Sugarcane bacilliform virus promoter confers gene expression preferentially in the vascular bundle and storage parenchyma of the sugarcane culm

“…The SCBV21:EYFP /pSK vector was produced by cloning the Sal Ι/ Nco Ι-released SCBV21 fragment of SCBV21 /pGEM-T as a transcriptional fusion with the EYFP gene in the Sal Ι/ Nco Ι-digested CaMV 2×35S: EYFP -NOS/pSK (pBluescript) vector [41], replacing the CaMV 2×35S promoter. The Pr4:EYFP /pSK construct was assembled by cloning the Hin dШ/ Nco Ι-released Pr4 fragment of Pr4:GUS /pUC19 (Invitrogen, Carlsbad, CA, USA) as a transcriptional fusion with EYFP into the Hin dШ/ Nco Ι-digested EYFP -NOS/pSK, replacing the CaMV 2×35S promoter.…”

“…The Pr4:EYFP /pSK construct was assembled by cloning the Hin dШ/ Nco Ι-released Pr4 fragment of Pr4:GUS /pUC19 (Invitrogen, Carlsbad, CA, USA) as a transcriptional fusion with EYFP into the Hin dШ/ Nco Ι-digested EYFP -NOS/pSK, replacing the CaMV 2×35S promoter. The 35S :EYFP -NOS/pSK vector was constructed by cloning the Hin dШ/ Bam HΙ-released CaMV 35S fragment from pBI221 (Clontech, Takara Bio USA, Inc., Mountain View, CA, USA) as a transcriptional fusion with the EYFP gene in the Ubi1 : EYFP -NOS/pSK vector [41] after digestion with two sets of restriction enzymes, Bam HΙ and Eco RΙ, and Eco RΙ and Hin dШ, replacing the Ubi1 promoter.…”

Genetic Engineering of Saccharum

“…To address this problem, vector-free minimal transgene cassettes (MGC) have been used to eliminate the use of unnecessary DNA and to produce transgenic plants with low copy number (Lowe et al 2009 ;Zhao et al 2007 ) . In a recent study in sugarcane, an increase of up to 160-fold in the level of transient expression of the EYFP reporter gene has been achieved by using a double terminator MGC (Beyene et al 2011 ). The utility of such system needs to be further tested in sugarcane stable transformants.…”

“…Reporter/scorable markers such as b -glucuronidase (GUS), GFP, yellow fl uorescent protein (EYFP), luciferase (LUC), and maize anthocyanin (ANT) regulatory elements have been used for the development and optimization of transformation to allow visual selection of transient and stable integrations (Arencibia et al 1995 ;Bower and Birch 1992 ;Bower et al 1996 ;Elliott et al 1998 ) , functional analysis (Braithwaite et al 2004 ;Damaj et al 2010 ;Gallo-Meagher and Irvine 1993 ;Liu et al 2003 ;Wei et al 2003 ) , subcellular targeting studies (Gnanasambandam and Birch 2004 ;Gnanasambandam et al 2007 ) , dissection of transgene silencing (Birch et al 2010a ) , and determination of terminator ef fi ciencies of constructs (Beyene et al 2011 ).…”

Transcriptome Analysis and Functional Genomics of Sugarcane

“…One potential problem with this strategy has been the reported propensity of sugarcane to silence transgenes ), a property mooted to be related to its ploidy. Although the reasons underlying transgene silencing in sugarcane are not completely understood, there are efforts underway to develop design principles for transgene constructs that may reduce its impact (Beyene et al 2010). In addition, the first reports of functional tissue-specific promoters from sugarcane, directing vascular expression, have opened the possibility of targeting transgene expression to particular cell types to study function (Damaj et al 2010a).…”