Ranunculaceae Class C Mads-box Gene Cloning And Expression

Flowers play an important role in the species reproduce and the formation of a new specie as the reproductive organs of angiosperms, The architecture of angiosperm flowers is complex and diverse. A considerable number of phylogenetic analyses have demonstrated that although the'ABCDE'classes of MADS-box genes possesses conserve functions in determining the floral development, they have undergone extensive gene duplication events and subsequent functional divergence during the evolution. Gene duplication and diversification which may lead to subfunctionalization or neofunctionalization can provide the raw material for the innovation of new morphologies and diversity of the flower in the angiosperm.However, how did the duplicate genes of'ABCDE'class MADS-box gene control the morphological diversity of flowers in angiosperms and what kind of influences in the flowers'formation and evolution are still unclear. But the plants of Ranuculaceae seems as a great group to exploring these problems, for that the diversity flowers in Rnuculaceae possess a rich mutant type flowers which include almost the whole morphological flowers of angiosperms. The mutants of petals in this family are especially interesting. Previous research found the formation of petals is highly correlated with the expression of B class of MADS-box genes especially the AP3-3 gene in Ranuculuaceae. By the way, previous morphology research found the petals of Ranuculuaceae are derived from the stamens. So we guess the C-classes MADS-box genes also involved in the initiation of the petal in Ranuculuaceae.To assess whether homologues of C class MADS-box gene in Ranuculuaceae may play a role in shaping the petal, we separate several C-class genes from 8 species which located in the different phylogenetic clades in Ranuculuaceae, and the detail shows that all the species possess two copies, which we named AG1 and AG2.the results implies that there is a gene duplications occurred before the original of Ranuculuaceae, which is consistent with the previous studies. At the same time, we suspect that the diversity of duplicate genes may provide raw materials for the evolution of the petals in the flowers of angiosperms. The expression pattern about the homologues of C-class genes in the representative species of Ranuculaceae is done by the In situ hybridization. The results indicate that the two copies in the Thalictrum aquilegiifolium which possess no petals have the same expression patterns; both are expressed in the stamens and carpals, which means they retain the original functions of their ancestor. However, in the closely related Leptopyrum fumarioides which produces petals, the two AG subfamily genes have the different expressions. LefuAG2 is expressed only in the carple primordium and the middle of the mature carpals, which means that this copy may lose the function in stamen identity,and we regard this as a kind of gene Subfunctionalization; but LefuAGlis not only expressed in the stamen and carpel, it also expressed in the petal primordium.we also detecte this gene in the lateral development of the petals.these results provide another evidence for the theory of that the petal of Ranuculaceae are derived from stamens.In conclusion, we think that the structures and functions of C-class MADS-box genes in Ranuculaceae are conserved by the evolutionary of the angiosperms. But the functional diversity and the change of expression patterns also occurred by the gene duplications and selections. Finally, the different copies acquired one or more new functions which may play a crucial role in petal development. We may conclude that the mechanism for the floral development may like this:1) C-class MADS-box genes are expressed in the petals, this may caused by the residue of C-class gene functions along the long evolution in which the outer stamens are transformed into petals, and finally made the stamens primodium developed into petals or organs between stamen and petal. Thus, the result further provides an evidence for the theory which admits that petals are developed from stamens.2) In the groups which possess no petals, the C-class genes will express their initial functions like determining the reproductive organs,and finally the primordium may develop as a stamen under the control of these genes while in the other groups which have petals, the C-class MADS-box genes may lose their original functions in the outer whole of the stamen primordium, and gradually lost their functions in determining the petals,in the meantime, the A and B function MADS-box genes especially the expressions of AP3-3 genes will control the development of petals.