Efficient gene tagging in Arabidopsis thaliana using a gene trap approach

Abstract

Large quantities of DNA sequence information about plant genes are rapidly accumulating in public databases, but to progress from DNA sequence to biological function a mutant allele for each of the genes ideally should be available. Here we describe a gene trap construct that allowed us to disrupt transcribed genes with a high efficiency in Arabidopsis thaliana. In the T-DNA vector used, the expression of a bacterial reporter gene coding for neomycin phosphotransferase II (nptII) depends on the in vivo generation of a translation fusion upon the T-DNA integration into the Arabidopsis genome. Analysis of 20 selected transgenic lines showed that 12 lines are T-DNA insertion mutants. The disrupted genes analyzed encoded ribosomal proteins (three lines), aspartate tRNA synthase, DNA ligase, basic-domain leucine zipper DNA binding protein, ATP-binding cassette transporter, and five proteins of unknown function. Four tagged genes were new for Arabidopsis. The results presented here suggest that gene trapping, using nptII as a reporter gene, can be as high as 80% and opens novel perspectives for systematic gene tagging in A. thaliana.

Figure 1

A schematic drawing of the T-DNA from the pNPTARP binary vector. The left and right borders of the T-DNA are indicated as triangles. The arrowed lines below the T-DNA map indicate sequences derived from the arp gene or the nptII gene, which lacks the first eight amino acids. Boxed regions are exons. The arp gene exons are numbered and the nptII gene region is shaded. Appropriate frames are indicated as f1, f2, and f3. At the f3 site is an in-frame fusion of the arp second exon-coding frame with the coding region of nptII. At this position a BamHI site was generated. The beginning of the arp-coding frame with the initiating translation ATG codon is indicated as f2, and a 24-bp downstream frame shift (indicated in brackets) was introduced by deleting a SacI site. The frame shift results in an arp–nptII fusion, the frame of which begins at the f1 site, but lacks an ATG start codon. If the T-DNA integrates into the Arabidopsis genome by homologous recombination with the arp gene over 420-bp upstream and 2.4-kb downstream sequences from nptII (areas indicated by black bars below T-DNA), the nptII protein can be synthesized from the arp first ATG codon. In this case, the SacI site will be derived from a genomic arp gene and detection of the 420-bp SacI–BamHI fragment on a DNA gel blots can be used to identify homologous recombination events. Alternatively, before stable integration into the Arabidopsis genome, the T-DNA may undergo deletions, from 570 bp to reach the f1 site and up to 1,130 bp to reach the nptII-coding region, as indicated by waved lines below the map. In this case the expression of nptII may occur as a result of T-DNA integration into Arabidopsis-expressed genomic regions, given that an in vivo fusion protein will be synthesized in transformed cells as result of either direct exon-exon integrations or after splicing of the generated chimeric intron.

Figure 2

DNA gel blot analysis of the transgenic Arabidopsis lines. DNA was extracted from heterozygous plants (+/−) after the third backcross of the indicated lines and from a wild-type Arabidopsis C24 (+/+) used for crosses. For the line SK1-N2, DNA extracted from six albino plants (−/−) segregating in a self-fertilized progeny was analyzed additionally. DNA was digested with PstI or HindIII; fragments were separated by agarose gel, blotted onto a nylon membrane, and hybridized with DNA probes made from cDNA fragments encoded by the respective T-DNA-tagged genes. In DNA from transgenic plants, new hybridizing fragments can be detected in addition to fragments with a wild-type mobility. The albino plants from the progeny of SK1-N2 appear to be homozygous for the T-DNA insertion and completely lack a 2.4-kb fragment.

Figure 3

dal1 mutant of Arabidopsis. (A and B) Two-month-old dal1 mutant plants are shown. Mutant plants from the same seed stock were grown in vitro in six Petri plates, but only plants from a single plate began to develop greenish young leaves (B). The only apparent difference between plants from these different plates is that in B plants are vitrified. The ultrastructure of plastids from mutant plants (D and F) compared to that of the wild type (C and E). Plants were either grown on agar-solidified Murashige and Skoog medium supplemented with 20 g/liter of sucrose (C and D) or grown in the same, but liquid medium on a gyratory shaker (E and F). In plastids of the dal1 mutant a membranous structure resembling thylakoids can be observed (F, but not in D). General morphology of the leaf cells for dal1 (H and J) compared to that of wild type (G and I). Young leaves of the plants grown on agar plates (G and H) or fully expanded leaves of plants grown in a liquid medium (I and J) were sectioned after imbedding into vinylcyclohexene dioxide (Spurr mixture). In the mutant, no palisade parenchyma cells developed and cells are more rounded. [Bars = 2 μm (C–F), 50 μm (G–H), and 100 μm (I–J).]