Expression Pattern And Functional Analyses Of B-function MADS-box Genes In Camellia Japonica

The economic value of flowers is reflected in their unique appearance and function.The classical ABC model,an early study of florals,illustrates the regulatory mechanism of floral development.A typical flower in higher plants can be divided into four whorls,which are regulated by different A-,B-,and C-type genes.The majority of ABC genes has the MADSbox domain.And the B class genes of the ABC model are the key genes for specifying the petals and stamens.The B class genes formed two branches of APETALA3(AP3)and PISTILLATA(PI)through early genome replication,and then AP3 developed into eu AP3,Paleo AP3 and TM6 three lineages.Particularly,the mechanism of the regulation of double flower formation is incomplete,though the key genes of floral development are identified.Here,we took the Camellia japonica L.as experimental materials and cloned four B class genes that were homologous to AP3 and PI.We named them as GLOBOSA1(CjGLO1),GLOBOSA2(CjGLO2),DEFICIENS(CjDEF)and TOMATO MADS BOX GENE6(CjTM6).We collected the floral organs of four types of camellia double flower,including formal double,semi-double,peony double,and anemone double.We found that the expression of class B genes was detected in all whorls,and is the highest in inner petals.The m RNA in situ hybridization experiments showed that CjDEF was mainly expressed in stamens,petals and carpels at the early stage.At the late stage,it was mainly expressed in petals and stamens.Then,we successfully transferred four class B functional genes into Nicotiana benthamiana by the Agrobacteria-mediated leaf disc method.The heterotopic expression of class B genes led to the phenotype of smaller petals,malformed petals,or even missing petals;and the stamens were reduced and the filaments were not elongated.Through the scanning electron microscopy(SEM)experiments on the flower organs of strong phenotype,we found that the adaxial cells of transgenic petals were smoother in general,and the morphology of cells was irregular;while the abaxial cells of normal petals were bulging,and the morphology of cells was nearly round.Although the morphology of transgenic distal axial cell was not different from that of normal petals,yet the structure similar to stomata appeared on the distal axial plane of the transgenic plant.The cell morphology of flower tubes and sepals did not change.The petal identity did not change,despite the petals of transgenic were changed.The quantitative PCR experiments of transgenic and endogenous genes were carried out on the mutant flower organs.The results showed that the B class genes were significantly upregulated in the strong phenotypic transgenic lines,but the expression of the B class genes of endogenous tobacco was down-regulated.Subcellular localization analysis showed that all four class B functional genes were localized in the nucleus.And the analysis by the Bimolecular Fluorescent complementarity experiments showed that CjDEF interacted with CjGLO1 and CjGLO2,and CjTM6 interacted with CjGLO1 and CjGLO2.These results indicate that the four class B functional genes have typical characteristics of class B functional genes and may play an important role in flower organ development.In our study,we show that four class B functional genes,CjGLO1,CjGLO2,CjDEF and CjTM6,are important factors affecting petal formation in Camellia,and verify the proteinprotein interaction relationship among class B genes.The results indicated that the class-B genes maybe take a replication and functional change and the Variation of Camellia flower type may be related to the regulation of the expression pattern of class B genes,which provided a theoretical basis for the genetic selection and breeding of ornamental traits of Camellia.