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

Abstract

The selection of transcription terminators (TTs) for pairing with high expressing constitutive promoters in chimeric constructs is crucial to deliver optimal transgene expression in plants. In this study, the use of the native combinations of four polyubiquitin gene promoters and corresponding TTs resulted in up to >3-fold increase in transgene expression in maize. Of the eight polyubiquitin promoter and TT regulatory elements utilized, seven were novel and identified from the polyubiquitin genes of Brachypodium distachyon, Setaria italica, and Zea mays. Furthermore, gene expression driven by the Cassava mosaic virus promoter was studied by pairing the promoter with distinct TTs derived from the high expressing genes of Arabidopsis. Of the three TTs studied, the polyubiquitin10 gene TT produced the highest transgene expression in maize. Polyadenylation patterns and mRNA abundance from eight distinct TTs were analyzed using 3'-RACE and next-generation sequencing. The results exhibited one to three unique polyadenylation sites in the TTs. The poly(A) site patterns for the StPinII TT were consistent when the same TT was deployed in chimeric constructs irrespective of the reporter gene and promoter used. Distal to the poly(A) sites, putative polyadenylation signals were identified in the near-upstream regions of the TTs based on previously reported mutagenesis and bioinformatics studies in rice and Arabidopsis. The putative polyadenylation signals were 9 to 11 nucleotides in length. Six of the eight TTs contained the putative polyadenylation signals that were overlaps of either canonical AAUAAA or AAUAAA-like polyadenylation signals and AUGAAU, a top-ranking-hexamer of rice and Arabidopsis gene near-upstream regions. Three of the polyubiquitin gene TTs contained the identical 9-nucleotide overlap, AUGAAUAAG, underscoring the functional significance of such overlaps in mRNA 3' end processing. In addition to identifying new combinations of regulatory elements for high constitutive trait gene expression in maize, this study demonstrated the importance of TTs for optimizing gene expression in plants. Learning from this study could be applied to other dicotyledonous and monocotyledonous plant species for transgene expression. Research on TTs is not limited to transgene expression but could be extended to the introduction of appropriate mutations into TTs via genome editing, paving the way for expression modulation of endogenous genes.

Keywords: Poly(A) signals; gene silencing; maize; polyadenylation sites; polyubiquitin gene promoter; transcription terminator; transgene expression.

Figure 1

Schematic representation of assembled T-DNA cassettes: promoters and TTs are depicted as green arrows and yellow boxes, respectively. Distinct RE combinations were tested using the yfp (A) and cry34Ab1 (B) reporter genes as depicted by pink and blue boxes, respectively. The T-DNA backbone and the selection marker, aad-1, present at the 3′ end of the cassettes are not shown in the schematic. The numbers on the left of the cassettes denote the construct numbers. The numbers on the top left of the TT boxes depict coordinates corresponding to the 3′ RACE sequencing data and that on the top right of the boxes denote the total length of the TTs. The red arrow on top of the reporter gene box displays the oligo position for 3′ RACE analysis.

Figure 2

Transgene expression analysis of mRNA and protein in T₁ plants containing the yfp and cry34Ab1 reporter genes. qRT–PCR analysis of yfp (A) and cry34Ab1 (B) for relative mRNA transcript levels in V6 leaves of transgenic maize plants. The same plants were used to quantitate protein accumulation for yfp (C) and cry34Ab1 (D). All plants were derived from the hemizygous T₁ plants of distinct transgenic events. For each construct, the data points were collected from three to five independent transgenic events (lines) and minimum 10 sibling plants per event at V6. Data are represented as mean ± SD of the combined data points for each construct, and differences were analyzed with Student’s t-test. The comparison showing a significant difference is indicated (p<0.05). The negative control construct (121 and 746) transgenic plants when used with Figure 1B and Figure 1A group of transgenic plants, respectively, did not show any mRNA or protein expression.

Figure 3

3′ RACE sequence mapping profiles of mRNA extracted from V6 maize leaves at the T₁ generation. Two independent events per construct were used for 3′ RACE analysis. mRNA poly(A) sites and its abundance reflected in individual peaks are shown for distinct TTs. On the X-axis, the position 1 starts at the last base of the reporter gene. The Y-axis indicates the normalized reads number per million (RPM) of the 3′ RACE deep sequencing read end (after trimming poly (A) mapping for each position). The peak of poly(A) site representing mRNA abundance is determined using a cut-off of 10,000 RPM. For each construct, two individual samples were sequenced, and similar results were observed. The representative data is shown here in the sequence mapping profiles of up to 335 nt. (A) Comparison of constructs containing the StPinII TT (SiUbi2+PinII; BdUbi1-C+PinII, BdUbi1+PinII), SiUbi2 TT (SiUbi2+SiUbi2), BdUbi1 TT (BdUbi1+BdUbi1) and BdUbi1-C TT (BdUbi1-C+BdUbi1-C). (B) Comparison of StPinII TT (ZmUbi1+StPinII) and ZmUbi1 TT (ZmUbi1+ZmUbi1). (C) Comparison of AtELF1 TT (CsVMV+AtELF1), AtUBC9 TT (CsVMV+AtUBC9) and AtUbi10 TT (ZmUbi1+AtUbi10). The number indicated by the black triangle represents the position of the highest peak (highest mRNA abundance) for that construct.

Figure 4

DNA sequence of distinct TTs deployed in constructs of transgenic maize. The first bp in the sequence is the last bp of the reporter gene and coordinate 1 of the diagrams in Figure 3. The underlined sequences were present between the reporter gene coding sequence and the TTs. The turquoise, green and yellow highlighted sequences depict PPSGs and overlapping hexamers as shown in Table 1, major poly(A) sites representing most abundant mRNA as shown in Figure 3, and non-major poly(A) sites as shown in Figure 3, respectively. The letters in red and blue font are putative FUE motifs and mRNA stability motifs (e.g., CGTGTCTT of StPinII), respectively, that were previously described (Supplementary Table S2). If two motifs overlapped, only one of the motifs is shown.