Rice gibberellin-insensitive dwarf mutant gene Dwarf 1 encodes the alpha-subunit of GTP-binding protein

Abstract

A rice Dwarf 1 gene was identified by using a map-based cloning strategy. Its recessive mutant allele confers a dwarf phenotype. Linkage analysis revealed that a cDNA encoding the alpha-subunit of GTP-binding protein cosegregated with d1 in 3,185 d1 segregants. Southern hybridization analysis with this cDNA as a probe showed different band patterns in several d1 mutant lines. In at least four independent d1 mutants, no gene transcript was observed by Northern hybridization analysis. Sequencing analysis revealed that an 833-bp deletion had occurred in one of the mutant alleles, which resulted in an inability to express GTP-binding protein. A transgenic d1 mutant with GTP-binding protein gene restored the normal phenotype. We conclude that the rice Dwarf 1 gene encodes GTP-binding protein and that the protein plays an important role in plant growth and development. Because the d1 mutant is classified as gibberellin-insensitive, we suggest that the GTP-binding protein might be associated with gibberellin signal transduction.

Figure 1

Phenotypes of d1 mutant, Daikoku. In each photograph, Daikoku is on the left and Nipponbare (wild-type variety) is on the right. (A) Plant height. (B) Panicle. (C) Seeds.

Figure 2

High-resolution RFLP linkage map and physical map of the d1 locus. The vertical bar represents the RFLP markers, and the numbers of recombinants are indicated under the linkage map. Genetic distances between adjacent markers are shown in parentheses. □, End-fragment DNA of YAC clone; ○, RFLP markers or YAC end-fragment DNA contained in the YAC clones (Y number) and PAC clones (P number).

Figure 3

(A) Genomic Southern hybridization analysis of nine d1 mutants using S5933 as a probe. Lanes: 1, λ/HindIII; 2, Nipponbare; 3, Kasalath; 4, FL2; 5, HO532; 6, HO533; 7, HO537; 8, HO538; 9, HO541; 10, HO552; 11, CM382; 12, CM1792; 13, λ/HindIII. Three micorgrams of total DNA were digested with HindIII. (B) Northern hybridization analysis using S5933 (1) or S14002 (2) (actin) as a probe. Lanes: 1, Nipponbare; 2, Kasalath; 3, FL2; 4, HO532; 5, HO541; 6, HO552.

Figure 4

Complementation test. (A) Transgenic plants of d1 mutant (HO541). (1) Left, transgenic plant with cosmid vector; right, transgenic plant with cosmid clone (C6) containing D1 genomic region. (2) Left, seeds of transgenic plant with cosmid vector; right, seeds of transgenic plant with cosmid clone (C6). (B) Detection of integrated D1 gene in transgenic plants. Lanes: 1, marker (ϕX174/HaeIII); 2, Nipponbare (normal); 3, HO541 (d1 mutant); 4, cosmid clone (C6); 5–8, transgenic plants with cosmid vector in HO541 (phenotypes were all dwarf); 9–12, transgenic plants with cosmid clone C6 (phenotypes were all normal); 13, marker (ϕX174/HaeIII).