Complementation of plant mutants with large genomic DNA fragments by a transformation-competent artificial chromosome vector accelerates positional cloning

Abstract

To accelerate gene isolation from plants by positional cloning, vector systems suitable for both chromosome walking and genetic complementation are highly desirable. Therefore, we developed a transformation-competent artificial chromosome (TAC) vector, pYLTAC7, that can accept and maintain large genomic DNA fragments stably in both Escherichia coli and Agrobacterium tumefaciens. Furthermore, it has the cis sequences required for Agrobacterium-mediated gene transfer into plants. We cloned large genomic DNA fragments of Arabidopsis thaliana into the vector and showed that most of the DNA fragments were maintained stably. Several TAC clones carrying 40- to 80-kb genomic DNA fragments were transferred back into Arabidopsis with high efficiency and shown to be inherited faithfully among the progeny. Furthermore, we demonstrated the practical utility of this vector system for positional cloning in Arabidopsis. A TAC contig was constructed in the region of the SGR1 locus, and individual clones with ca. 80-kb inserts were tested for their ability to complement the gravitropic defects of a homozygous mutant line. Successful complementation enabled the physical location of SGR1 to be delimited with high precision and confidence.

Figure 1

Physical map of pYLTAC7. (A) The map shows the location of some sites for endonucleases that cleave the molecular once or twice. LB and RB, left and right borders, respectively; OD, overdrive sequence; Pnos, promoter of the nopaline synthase gene; HPT, coding region of the hygromycin phosphotransferase gene; nos 3′, polyadenylation signals of the nopaline synthase gene; KanR, kanamycin-resistance gene (NPT1). The complete sequence of the vector is available in the GenBank database (accession no. AB020028). (B) Sequence of the cloning-site region upstream of the sacB gene. The primer sets (R1, R2, R3, L1, L2, and L3) are designed for isolation of end fragments of the inserted DNA by thermal asymmetric interlaced PCR (TAIL-PCR) (22).

Figure 2

Stability of two TAC clones in E. coli and A. tumefaciens. TAC plasmid DNA of two independent clones were isolated from E. coli (DH10B) and were used for transformation of A. tumefaciens C58C1(MP90). The plasmid DNA in the Agrobacterium host was transferred back to the E. coli host. Digestion of plasmid DNA in each step is shown in this figure. (Left) A TAC clone digested by HindIII. (Right) Another TAC clone digested by PstI. M, molecular markers.

Figure 3

Transgenes in Arabidopsis plants transformed with TAC clones. (A) A. thaliana ecotype WS was transformed with TAC clones carrying either 40-kb (lanes 1–20) or 80-kb (lanes 21–36) genomic DNA fragments of ecotype Columbia. The sacB gene of 36 transgenic plants (hygromycin-resistant plants) was checked by PCR (Fig. 1). C+ and C−, positive (a TAC clone) and negative (untransformed plant) controls, respectively. (B) Transgenic lines transformed with a 75-kb TAC clone was self-crossed, and then the resulting T₂ plants were analyzed by genomic Southern experiments. Genomic DNAs of transgenic and untransformed (negative control) plants were digested with I-SceI and hybridized with a HPT gene probe. The hybridized bands (lanes 3, 5, and 6) are shown by the arrow on the right. No hybridization band corresponding to the I-SceI fragment is seen in lanes 2 and 4. Lane 1, plasmid DNA digested with I-SceI; lanes 2–6, genomic Southern blotting of DNAs from untransformed plants (lane 2) and T₂ lines (lanes 3–6). (C) The progenies of a transgenic line transformed with a 45-kb TAC clone were analyzed. Genomic DNAs of a T₂ line (lane 2) and its T₃ progenies (lanes 3 and 4) were digested with I-SceI and hybridized with the HPT gene probe. Lane 1, plasmid DNA digested with I-SceI; lanes 2–4, genomic Southern blotting of DNA digested with I-SceI.

Figure 4

Complementation of the sgr1 mutation with large TAC clones of wild type. (A) The SGR1 locus was covered contiguously by TAC clones (13E18, 3P22, 20D10, 5I12, and 25N15) carrying large (ca. 80-kb) genomic DNA fragments of A. thaliana Columbia ecotype that were isolated by using two DNA markers, CDC2B and KSAP3. (B) Segregation of the T₂ family seedlings of the transformed line E for gravitropic responses. Seedlings were grown in darkness for 3 days after germination with the plate setting as the direction of gravity indicated by g-1. The plate then was turned by 90° as indicated by g-2, and the seedlings were grown in darkness for 24 hr. About 75% of seedlings showed distinct negative gravitropic curvature in hypocotyls as wild type, whereas the remaining (marked by arrows) did not show gravitropic curvature at all (sgr1 mutation phenotype).