Development of an activation tagging system for maize

Abstract

Activation Tagging, distributing transcriptional enhancers throughout the genome to induce transcription of nearby genes, is a powerful tool for discovering the function of genes in plants. We have developed a transposable element system to distribute a novel activation tagging element throughout the genome of maize. The transposon system is built from the Enhancer/Suppressor (En/Spm) transposon system and uses an engineered seed color marker to show when the transposon excises. Both somatic and germinal excision events can be detected by the seed color. The activation tagging element is in a Spm-derived non-autonomous transposon and contains four copies of the Sugarcane Bacilliform Virus-enhancer (SCBV-enhancer) and the AAD1 selectable marker. We have demonstrated that the transposon can give rise to germinal excision events that can re-integrate into non-linked genomic locations. The transposon has remained active for three generations and events displaying high rates of germinal excision in the T2 generation have been identified. This system can generate large numbers of activation tagged maize lines that can be screened for agriculturally relevant phenotypes.

Keywords: activation tagging; enhancer; insertional mutagenesis; transposable elements.

Figure 1

Schematic representation of the T‐DNA in pEPS3004. Ubi::Spm TPase represents the maize Ubiquitin 1 promoter driving the Spm transposase gene. GLOB1 Promoter represent the maize Globulin 1 promoter. C1 and B‐peru represent the coding sequences of the maize anthocyanin transcription factors C1 and B‐peru. 5′TIR and 3′TIR represent the terminal inverted repeats of the Spm transposon. 4xSCBV represent four copies of the SCBV enhancer sequence. OsActin:AAD1 represents the rice actin promoter driving expression of the AAD1 herbicide tolerance gene. The 5′TIR: 4xSCBV: OsActin:AAD1: 3′TIR is the non‐autonomous transposable element located in the 5′ UTR of the GLOB1 Promoter: B‐peru gene

Figure 2

Examples of transient assays showing excision of transposon from pEPS3004. The purple sectors indicate site of excision

Figure 3

Kernel phenotypes showing (a) purple sectors and (b) purple kernels

Figure 4

Ear phenotypes from T0 plants. (a) An ear with all yellow kernels. (b) An ear showing ~50% yellow kernels and ~50% yellow kernels with purple sectors. (c) An ear showing ~50% yellow kernels and ~50% solid purple kernels. (d) An ear showing ~50% yellow kernels and ~50% yellow kernels with purple sectors plus a few solid purple kernels

Figure 5

Distribution of ear phenotypes in pEPS3004 events in T1 (a), T2 (b) and T3 (c) generations

Figure 6

Distribution of T1 ear phenotypes in events with different copy number as determined by DNA gel blot analysis. The number of samples in each category are listed above the bars

Figure 7

Frequency of putative germinal excision in events in the T1 to T2 (a) and T2 to T3 (b) generations

Figure 8

Identification of junction read pairs and individual reads to characterize launching and tag sites. (a) Schematic of sequence analysis of the pEPS3004 T‐DNA prior to and after excision of the transposon. The brown bar represents chromosomal sequences where the T‐DNA integrated (designated on Chromosome X) after excision and re‐integration the transposons integrate on Chr Y and ChrZ. (b) Schematic of sequence analysis of the pEPS3004 insert and the non‐autonomous transposon containing the Enhancer and Herbicide Resistance (HR) marker after transposon excision. (c) The genomic location of the T‐DNA is determined (blue reads which map to chrX. The genomic location of the site of transposon re‐insertion is determined by identifying reads that span the junctions between the transposon and insertion site (light blue and orange reads to chrY, chrZ)

Figure 9

Insertion of the Spm‐derived Activation Tagging transposon occurs independent of the launch site. Launch sites are designated by circles and insertion sites are designated by triangles. The patters designate transposons from different launch sites