Open AccessIsolation of the Arabidopsis GA4 locus.
Author(s) -
Hui-Hwa Chiang,
Inhwan Hwang,
Howard M. Goodman
Publication year - 1995
Publication title -
the plant cell
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.324
H-Index - 341
eISSN - 1532-298X
pISSN - 1040-4651
DOI - 10.1105/tpc.7.2.195
Subject(s) - biology , arabidopsis , mutant , genetics , complementation , southern blot , microbiology and biotechnology , genomic dna , ethyl methanesulfonate , silique , dna , gene
Progeny from a transgenic Arabidopsis plant generated by the Agrobacterium root transformation procedure were found to segregate for a gibberellin (GA)-responsive semidwarf phenotype. Complementation analysis with genetically characterized GA-responsive mutants revealed that the transgenic plant has an insertional mutation (ga4-2) that is an allele of the ga4 locus. The semidwarf phenotype of ga4-2 is inherited as a recessive mutation that cosegregates with both the T-DNA insert and the kanamycin resistance trait. DNA gel blot analysis indicated that the insertion site contains a complex T-DNA unit. A genomic library was constructed with DNA from the tagged ga4 mutant; a DNA clone was isolated from the library that flanks the T-DNA insert. The plant sequence isolated from this clone was used to isolate the corresponding full-length genomic and cDNA clones from wild-type libraries. DNA sequence comparison of the clones to the existing data bases suggests that they encode a hydroxylase. This conclusion is in agreement with a biochemical study that indicated that the ga4 mutant is deficient in 3 beta-hydroxylase in the GA biosynthetic pathway of Arabidopsis. RNA gel blot analysis showed that the message is ubiquitously expressed in different tissues of Arabidopsis but most abundantly in the silique. Unexpectedly, a higher level of transcription was detected in the ethyl methanesulfonate-induced ga4 mutant, and this overexpression was repressed by treatment with exogenous GA.