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

Abstract

Key messageThe choice of promoter regulating the selectable marker gene impacts transformation efficiency, copy number and the expression of selectable marker and flanking genes in maize.AbstractViral 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.

Transcription Terminator-Mediated Enhancement in Transgene Expression in Maize: Preponderance of the AUGAAU Motif Overlapping With Poly(A) Signals

“…Two positive control constructs, ZmUbi1 promoter/yfp/StPinII TT (121) and ZmUbi1/cry34Ab1/StPinII TT (746), were assembled to use as benchmarks for gene expression evaluations. More details of yfp and aad-1 are described previously (Beringer et al, 2017). The aad-1 cassette was the same in all constructs.…”

“…Agrobacterium-mediated transformation of maize and plant growth conditions were described previously (Beringer et al, 2017). Thirty to forty T 0 plants were regenerated and assayed for yfp or cry34Ab1, and aad-1 copy number using quantitative PCR assays (see below).…”

“…Total DNA was extracted from the leaf samples using MagAttract ® DNA extraction kit (Qiagen, MD, USA) as per the manufacturer's instructions. Copy number assays for yfp and aad-1 were performed as described previously (Kumar et al, 2015;Beringer et al, 2017). The cry34Ab1 quantitative PCR assays were performed similarly except for the following details.…”

“…The invertase gene (Genebank Accession no. U16123.1) was used as an endogenous reference control, and the same primers and probes for this gene were used as described previously (Beringer et al, 2017). DNA was analyzed in a Roche LightCycler ® 480 System under the following conditions: 1 cycle of 95°C for 10 min; 40 cycles of the following 3-steps: 95°C for 10 seconds, 58°C for 35 seconds, and 72°C for 1 second; and a final cycle of 4°C for 10 seconds.…”

RNase If -treated quantitative PCR for dsRNA quantitation of RNAi trait in genetically modified crops

“…The binary vector was then transformed into Agrobacterium tumefaciens . The binary vector was transformed into maize via Agrobacterium -mediated transformation of immature embryos isolated from the inbred line, Zea mays c.v. B104 following the standard method [ 23 ].…”