A versatile transposon-based activation tag vector system for functional genomics in cereals and other monocot plants

Abstract

Transposon insertional mutagenesis is an effective alternative to T-DNA mutagenesis when transformation through tissue culture is inefficient as is the case for many crop species. When used as activation tags, transposons can be exploited to generate novel gain-of-function phenotypes without transformation and are of particular value in the study of polyploid plants where gene knockouts will not have phenotypes. We have developed an in cis-activation-tagging Ac-Ds transposon system in which a T-DNA vector carries a Dissociation (Ds) element containing 4x cauliflower mosaic virus enhancers along with the Activator (Ac) transposase gene. Stable Ds insertions were selected using green fluorescent protein and red fluorescent protein genes driven by promoters that are functional in maize (Zea mays) and rice (Oryza sativa). The system has been tested in rice, where 638 stable Ds insertions were selected from an initial set of 26 primary transformants. By analysis of 311 flanking sequences mapped to the rice genome, we could demonstrate the wide distribution of the elements over the rice chromosomes. Enhanced expression of rice genes adjacent to Ds insertions was detected in the insertion lines using semiquantitative reverse transcription-PCR method. The in cis-two-element vector system requires minimal number of primary transformants and eliminates the need for crossing, while the use of fluorescent markers instead of antibiotic or herbicide resistance increases the applicability to other plants and eliminates problems with escapes. Because Ac-Ds has been shown to transpose widely in the plant kingdom, the activation vector system developed in this study should be of utility more generally to other monocots.

Figure 1.

T-DNA of the activation-tagging Ac-Ds vector pSQ5. RB and LB, Right and left borders of the T-DNA; Ubi, maize ubiquitin 1 promoter; GFP, GFP gene as a negative selection marker; RFP, RFP (or DsRed) gene as selection marker for the Ds element; 4xEn, a tetramer of CaMV 35S enhancers; HPH, hygromycin phosphotransferase gene as plant transformation selection marker. The T-DNA is in the backbone of pCAMBIA-1300.

Figure 2.

GFP and RFP fluorescence assay of young seedlings (T₂) derived from pSQ5 transgenic rice plants. A, GFP and RFP assays and the scheme to determine GFP and RFP phenotype of each T₂ plant. B, A diagram showing a transposition event in the primary transformant (T₁) and its germinal transmission to T₂ population.

Figure 3.

Selection of stable Ds insertion lines and generation of Ds FSTs.

Figure 4.

Semiquantitative RT-PCR analysis of five Ds insertion lines. Left, Schematic representation of the Ds insertion and the CaMV 35S enhancers (4xEn) relative to the nearby genes. The map of each chromosomal region was based on the Rice GE genome database RiceGE Functional Genomics Database (http://signal.salk.edu/cgi-bin/RiceGE). Genes that were examined in semiquantitative RT-PCR are marked with dotted-line boxes. Right, RT-PCR analysis of activation-tagged genes. Rice Act1 transcript was amplified as control. WT, Wild-type rice ‘Nipponbare’. RT-PCR using leaf or root RNA was indicated. A, Line ADS247 was tagged by the activation-tagging Ds element 1,609 bp upstream of Os03g15050, which encodes the phosphenolpyruvate carboxykinase. B, Line ADS248 was tagged by a Ds 1,880 bp upstream of Os11g29490, which encodes a plasma membrane-type ATPase. C, Line ADS305 was tagged by a Ds 12.2 kb upstream of Os09g30439, which encodes a heat shock protein. D, Line ADS427 was tagged by a Ds 1,349 bp upstream of Os04g42840, which encodes a HEAT repeat family protein. E, Line ADS210 was tagged by a Ds 11.8 kb upstream of Os10g41410, which encodes the nucleoside diphosphate kinase 1. For each gene, the RT-PCR result was confirmed using two different pairs of specific primers and the result from using one pair of primers was shown. Each PCR experiment was repeated at least three times to confirm the result.