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. 2017 Apr;36(4):519-528.
doi: 10.1007/s00299-017-2099-y. Epub 2017 Feb 3.

Comparison of the impact of viral and plant-derived promoters regulating selectable marker gene on maize transformation and transgene expression

Jeffrey Beringer  1 , Wei Chen  1 , Russell Garton  2 , Nagesh Sardesai  1 , Po-Hao Wang  1 , Ning Zhou  1 , Manju Gupta  1 , Huixia Wu  3
Affiliations

Comparison of the impact of viral and plant-derived promoters regulating selectable marker gene on maize transformation and transgene expression

Jeffrey Beringer et al. Plant Cell Rep. 2017 Apr.

Abstract

The choice of promoter regulating the selectable marker gene impacts transformation efficiency, copy number and the expression of selectable marker and flanking genes in maize. Viral or plant-derived constitutive promoters are often used to regulate selectable marker genes. We compared two viral promoters, cauliflower mosaic virus (CaMV 35T) and sugarcane bacilliform virus (SCBV) with two plant promoters, rice actin1 (OsAct1) and maize ubiquitin 1 (ZmUbi1) to drive aryloxyalkanoate dioxygenase (aad-1) selectable marker gene in maize inbred line B104. ZmUbi1- and OsAct1-containing constructs demonstrated higher transformation frequencies (43.8 and 41.4%, respectively) than the two viral promoter constructs, CaMV 35T (25%) and SCBV (8%). Interestingly, a higher percentage of single copy events were recovered for SCBV (82.1%) and CaMV 35T (59.3%) promoter constructs, compared to the two plant-derived promoters, OsAct1 (40.0%), and ZmUbi1 (27.6%). Analysis of protein expression suggested that the viral promoter CaMV 35T expressed significantly higher AAD-1 protein (174.6 ng/cm2) than the OsAct1 promoter (12.6 ng/cm2) in T0 leaf tissue. When measured in T2 callus tissue, the two viral promoters both had higher expression and more variability than the two plant-derived promoters. A potential explanation for why viral promoters produce lower transformation efficiencies but higher percentages of low copy number events is discussed. In addition, viral promoters regulating aad-1 were found to influence the expression of upstream flanking genes in both T0 leaf and T2 callus tissue.

Keywords: Aad-1 and yfp genes; Copy number; Gene expression; Maize; Promoter choice; Transformation efficiency.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
The effect of different promoters regulating the selectable marker gene aad-1 on maize B104 transformation frequency. Calculated transformation frequency is the percentage of treated embryos which produced at least one viable shoot after selection on media containing the herbicide haloxyfop. To reduce variation, the transformation experiments for each repeat were carried out on the same day and all transfers used the same media lots
Fig. 2
Fig. 2
The effect of different promoters regulating the selectable marker gene aad-1 on copy number of both aad-1 and yfp genes. Leaf punches were taken from T0 shoots immediately prior to transplanting to soil. Events were categorized based on qPCR as containing a single copy, two copies, or three or more copies of the trangenes which is labeled complex
Fig. 3
Fig. 3
Effect of different promoters regulating the selectable marker gene on protein expression in callus derived from T2 embryos. All callus events were from embryos prescreened for YFP expression and are hemizygous for both transgenes. Protein was extracted from 50 to 100 mg of callus tissue using a PBS-based buffer, total soluble protein (tsp) was determined with the Pierce 660 nm protein assay, and ELISA was used to quantify AAD1 and YFP proteins using monoclonal antibodies. a, b AAD-1 and YFP (repectively) protein expression in T2 callus derived from constructs containing CaMV35T, OsAct1, SCBV and ZmUbi1 driving the aad-1 selectable marker gene. The centerline of the diamonds indicates the group mean. Expression data were from between 5 and 9 events per promoter and each data point represents a unique callus derived from a single embryo. Each event was represented by at least five and as many as 12 different calli. c AAD1 protein expression plotted by event and showing the callus to callus variability within each event
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