The Isolation and Functional Identification of MAwuAP3₁/2in Magnolia Wufengensis

Magnolia wufengensis is a newly discovered species in Magnoliaceae family.The variation of petal number ranging from9to32and its diversity of flower color,contributes an excellent material to study flower organ morphogenesis in plants andcarry comparative genetics research. The APETALA3（AP3） is a crucial class BMADS-box genes, which were firstly found and involved in specifying petal andstamen identity in Arabidopsis, whose homologs highly conserved in flowerdevelopment regulation of most dicotyledons and monocotyledons.Magnolia family is one the most ancient basal class in angiosperm with differentflower traits. In this paper, we took Homology-based cloning and Rapid-amplificationof cDNA ends （RACE） to isolate two genes, MAwuAP3₁/2of B-class gene, whichregulate stamen and petal development, combining with Semi-quantitative RT-PCRand Real-time fluorescence quantification PCR to specify the expression in time andtissue. Furthermore, the recover study of these two genes on Arabidopsis ap3-3mutant indicated the evolution and the main function in controlling Magnoliawufengensis flower development.The main results are as followings:（1） MAwuAP3₁/2cloning and expression analysis in Magnolia wufengensis:MAwuAP3₁/2, these B-class MADS-box genes involved in flower development hadbeen isolated from Magnolia wufengensis via the way of homology cloning andRACE technique. Protein sequence alignment and molecular phylogeny analysisdemonstrated a highly conserved paleoAP3motif was in the C-terminal regions of theMAwuAP3₁/2proteins, which also belongs to B-class paleoAP3evolution derivation;however, which lacks of the PI-derived motif, often appearing in AP3/DEF-like derivation.While the two MADS-box genes had9amino acid residue difference in K-zone and4aminoacid residue in C-zone.（2） Expression patern analysis of MAwuAP3₁and MAwuAP3₂from Magnoliawufengensis: semi-quantity RT-PCR displayed MAwuAP3₁or/and MAwuAP3₂only expressed in petals and stamens not in spires and pistils; RT-PCR uncovered indifference development stages, MAwuAP3₁maintained high level when petals andstamens growing rapidly, but MAwuAP3₂could be detected only during the initialstage of petals and stamens forming. Therefore, these two genes demonstrated similarexpression paterns in specifying tissue but in different time. （3） Function analysis of MAwuAP3₁/2: MAwuAP3₁/2was introducted intobinary expression vector Pbi121by CaMV35S promoter, and then transformed intoArabidopsis ap3-3mutant mediated by agrobacterium tumefaciens. Observed35S::MAwuAP3₁/2transgenic plants in homozygous ap3-3mutant throughStereopscopic microscopy techniques and Scanning electric photograph and indicatedthat both of the two genes patially recovered flower morphology of ap3-3, such asproducing filament-like organs in the third whorl and conversion of the second sepalsinto petals. Moreover, ectopic expression of MAwuAP3₁or/and MAwuAP3₂inAP3/ap3-3hybrid and in wild type,30%of the crops were increased1-3patals in thefirst blooming flower.From the above results, it’s approved that MAwuAP3₁/2highly conserved inregulating stamens and patals development, but varied with AP3homology genes inother plant family, which set up the theory foundation for AP3homology genesfunction evolution from basal group of angiosperm to core eudicots.