The Functional Identification Of MawuAG In Magnolia Wufengensis And Homologe Sequence Evolution Analysis Of Its Related Species

Magnolia wufengensis was a newly discovered species in recent yearsbelonging to Magnoliaceae family. Due to its variation of petal number ranging from9to32and its diversity of flower color, it became an excellent material to studyplant flower organ morphogenesis and carry comparative genetics research.AGAMOUS (AG) is a crucial class-C MADS-box gene in Arabidposis, which plays acritical role in promoting inflorescence meristems, regulating the development ofstamen and carpel as well as repressing class-A activity. Extensive research havebeen done about molecular mechanism involving controlling flower organmorphogenesis in model plant and staple corps but related research in regulatingflower development of ornamental plant is seriously lack, especially in basalangiosperms.Our study combined developmental biology and molecular biology to analyzethe expression pattern and biological function of AGAMOUS in flower developmentprocess, in order to discuss the molecular mechanism of flower formation inmagnolia wufengensis. Our conclusion is as following:(1) Cloning and expression analysis of MAwuAG from magnolia wufengensisA MADS-box gene MAwuAG involved in flower development has been isolatedfrom magnolia wufengensis via the way of homology cloning combined with RACEtechnique. Protein sequence alignment and molecular phylogeny analysissuggested that, MAwuAG is an AG homology in Arabidopsis, at the transcriptionalactivation domain of C-terminal exist two conserved motifs: AGⅠ andAGⅡ.Semi-quantitative RT-PCR analysis showed, MAwuAG transcriptional activitymainly concentrated on stamen and carpel but with low activity in flower perianthiawhile no-expression in leaves during different stages of flower development. Theanalysis of Northern blot is conforming to semi-quantity PCR.(2) Functional analysis of MAwuAG from magnolia wufengensisA sense expression vector of MAwuAG has been introduced into Arabidopsis viaagrobacterium-mediated method then screen positive transformation plants.Compared with wild type,35S::MAwuAG transgenic plants showed abnormalphenotype with petal converting into filament-like structure. However, stamen andcarpel develop normally, and sepal did not convert into capel-like structure.Moreover, the abscissions of the outer three floral whorls (sepals, petals, and stamens) were inhibited during development of the siliques, which were bumpy.And the seed set rate are lower than that of normal seeds in the wild type.(3) Phylogenetic relationships of magnolia wufengensis related speciesThe AGAMOUS homologs from39species in magnoliaceae family wereanalyzed to address phylogenetic questions in the family. The results showed that:Magnolia, the biggest genus in the family, is not monophyletic, and the associatesof Michelia are grouped with Magnolia subgenus Yulania, the Magnolia subgenusMagnolia are closely allied; a well-supported Manglietia clade is recognized.Based on the results above, we conclude that MAwuAG is a homology ofAGAMOUS in Arabidopsis with conserved function and it involved in regulation thedevelopment of stamen and carpel while did not develop the activity of repressingclass-A genes. Phylogenetic analysis inferred from AGAMOUS genes display thatM. wufengensis is grouped with Magnolia subgenus Yulania.