Characterization of gibberellin 20-oxidases in the citrus hybrid Carrizo citrange

CcGA20ox1 is a gene encoding a GA 20-oxidase, a gibberellin (GA) biosynthetic enzyme, previously isolated from the citrus hybrid Carrizo citrange (Citrus sinensis (L.) Osbeck x Poncirus trifoliata (L.) Raf.). Southern blot analysis of genomic DNA of Carrizo citrange with CcGA20ox1 suggested the presence in the hybrid of another gene encoding another GA 20-oxidase. A cDNA clone from this new gene (CcGA20ox2) was isolated using RNA from the other parent C. sinensis. CcGA20ox2 encoded a protein of 372 amino acids that showed 67.1% identify with CcGA20ox1, and its expression product catalyzed the in vitro conversion of GA(12) to GA(9), confirming that it corresponds to another active GA20ox. Amplification of genomic DNA and isolation of genomic clones of CcGA20ox1 and CcGA20ox2 revealed that the parental sources these genes in the hybrid were P. trifolicata and C. sinensis, respectively. The sequences of CcGA20ox1 and CcGA20ox2 showed that both genes contained two introns, which are also conserved in GA20ox genes of other species like Arabidopsis thaliana L., Pisum sativum L. and Solanum lycopersicum L. Determination of transcript levels in the Carrizo citrange hybrid by quantitative real-time polymerase chain reaction showed that CcGA20ox1 and expressed mainly in internodes, leaves and seeds, and CcGA20ox2 in flower buds and flowers at anthesis, with the genes having similar transcript levels in young developing fruits.