Details

Autor(en) / Beteiligte

• Singer, Stacy D

Titel

A functional analysis of KNOX genes and MIKCc-type MADS-box genes in the moss, Physcomitrella patens

Ort / Verlag

ProQuest Dissertations & Theses

Erscheinungsjahr

2005

Link zum Volltext

• ProQuest

Quelle

ProQuest Dissertations & Theses A&I

Beschreibungen/Notizen

• KNOX genes, a group of plant homeobox genes, are essential for the generation and/or maintenance of shoot apical meristems. MADS-box genes are involved in many ontogenetic processes including the differentiation of floral meristems and the determination of floral organ identities. Since flowers are the hallmark of angiosperms and all aerial parts of a flowering plant are derived from the shoot apical meristem, phylogenetic and functional analyses of members of these gene families from non-flowering plants should impart a more profound understanding of the origin and evolution of the various major land plant groups and the structures that characterize each of them. Physcomitrella patens is an attractive system for studying the functions of MADS-box and KNOX gene homologues, as in addition to its phylogenetically basal position among land plants, simple life cycle and similarities to higher plants, it is especially amenable to gene targeting. Gene knockout and knock-down approaches were used to obtain clues about the wild type functions of MADS-box and KNOX genes in P. patens. All single gene knockout strains and all combinations of double and triple gene knockout strains involving the three known P. patens KNOX genes (MKN1-3, MKN2 and MKN4) were generated, and their knockout status was verified at the molecular level. The knockout status of strains in which three P. patens MIKCc MADS-box genes (PPM1, PpMADS1 and PPM2) were disrupted either singly or as double knockouts was also confirmed. In addition, stable recombinants, created by transforming P. patens protoplasts with a vector including the PPM1 antisense cDNA controlled by a constitutive promoter (Krogan, 1999), were also examined at the RNA and DNA levels. Finally, the expression patterns of all MIKCc and KNOX genes considered here were studied. Phenotypic analysis of the various MKN knockout strains, as well as expression analysis of each of the MKN genes, indicated that class 1 KNOX genes in P. patens are involved in sporophyte development and probably function at least somewhat redundantly with one another. Conversely, no function could be ascribed to the class 2 KNOX gene, MKN1-3, either because there are redundantly acting genes in the moss genome or because this gene does not play an important role in P. patens development. Several PPM1 antisense strains exhibited a complex mutant phenotype, suggesting that MADS-box genes in P. patens are involved in a range of developmental functions including leaf morphology, moisture retention, gametangia formation and sporophyte development. Interestingly, while the majority of single and double MIKCc gene knockout strains possessed no noticeable phenotypic abnormalities, two PPM1 and two PPM2 single gene knockout strains exhibited phenotypes with characteristics in common with the PPM1 antisense phenotype. Because a number of transgenic P. patens strains in this study were observed to exhibit transgene silencing of the selectable marker gene, studies were undertaken to shed light on the mechanism underlying it. Molecular analyses of transgenic strains displaying transgene silencing indicated that methylation of the coding region of the selectable marker gene is involved.