Functional analysis of Arabidopsis proly 4-hydroxylase 7 T-DNA mutannts
2004
Radu, T. G. M.
The Arabidopsis genome sequence project facilitated the development of numerous reverse genetics approaches for the determination of gene function. Arabidopsis was used as a model system to investigate the role of plant prolyl 4- hydroxylases in plant growth and development. Prolyl 4-hydroxylases are members of the superfamily of 2-oxoglutarate-dependent dioxygenases and have an absolute requirement for Fe(II) as a cofactor, with dioxygen as a cosubstrate. Among the large family of prolyl 4-hydroxylases (P4Hs) encoded by the Arabidopsis genome, P4H7 is rapidly up-regulated in response to hypoxia, indicating a possible role i the triggerin mechanism of hvpoxic adaptation. The aim of the present study was to investigate the function of P4H7 through analyses of p4h7 T-DNA insertional mutants. The first step was the confirmation for the presence of T-DNA insertion. Two Arabidopsis p4h7 T-DNA mutants were identified, based on PCR tests, that had the insertion. Since the sequence of the insertion is known, primers complementary to the end of the insertion and the gene of interest were used for PCR screening. Having a confirmed prolyl 4-hydroxylase T-DNA mutant, the following step was to determine the effect on growth and development relative to the wild type. In the case of heterozygous plant progeny, the hypothesis used was that of having double recessive mutation (1/16 fits with the chi-square statistical test results. In the case of the homozygous p4h7C line, phenotypic analysis showed that the roots of p4h7C arc longer compared with the roots of the Arabidopsis wild type plants. Proteins containing 4-hydroxyproline are important structural components of plant cell walls. Further analyses are needed in order to investigate the role of proline hydroxylation in plant growth and development.
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