Functional Analysis Of MADS-box Genes Related To Floral Organ Development In Prunus Mume

Prunus mume, a traditional flower in China, possesses high ornamental value and profound cultural connotation. The morphologies of flower organs and the whole flower have varied greatly in P. mume during the long period of domestication and cultivation. However, the mechanisms that control flower organs development and morphologies is unavailable for P. mume. Previous studies have proved that MADS-box genes play key roles in flower development. However, little information on this gene family is available for P. mume. The number, classification, evolution, and roles of MADS-box genes in flower development of P. mume are still unknown. Therefore, the roles of the MADS-box genes played in P. mume flower development were investigated in this study. First, the genome-wide analyses of the MADS-box gene family in P. mume were performed. Then, ten genes related to flower development were cloned from P. mume’ Sanlun Yudie’ by RT-PCR. Their expression patterns were determined by quantitative RT-PCR; the yeast two-hybridization was employed to detect the protein-protein interactions between P. mume flower development related genes. All these results together with the phenotype changes in Arabidopsis over-expressing these genes were used to elucidate the roles of these genes. In addition, the mechanisms that control flower organs development in P. mume were also proposed. The main results of this study are as following:1.80 MADS-box genes were identified in P. mume,48 of them were type Ⅰ genes while 32 were type Ⅱ. Type Ⅰ genes can be divided into four subfamilies. Genes belonging to Mα, Mβ and Mγ subfamilies duplicated after the divergence of P. mume and Arabidopsis. However, MS subfamily genes exhibited similar gene structures and evolutionary patterns to type Ⅱ genes. Type Ⅱ genes can be classified into 12 subfamilies. The FLC subfamily was absent in P. mume and the six DAM genes might experience a genus-specific evolution process in Prunus. The MADS-box gene family might experience an evolution process from type Ⅱ genes to Mδ genes to Mα, Mβ and Mγ genes. Expression patterns based on transcriptom analysis revealed that the MADS-box genes of P. mume might regulate different organs’development and their roles were similar to their orthologous. The function of duplicated genes diverged after the duplication events.2. RT-PCR was employed to clone ten flower development related MADS-box genes from P. mume. The expression patterns of most P. mume MADS-box genes in different organs were similar to their orthologous genes but some genes were diverged. The transcript of PmAP1 can be detected only in sepals. The expression patterns of PmAP3 and PmAP3-2, which belonging to TM6 lineage and euAP3 lineage respectively, were similar to genes belonging to euAP3 lineage and TM6 lineage, respectively. E-class genes regulate flower and fruit development in different plants but the function of different members might diverge among species. In general, P. mume flower development related MADS-box genes were up-regulated during the flower differentiation in different cultivars. PmAP1, PmFULl, PmAG and PmSEP4 may involve in the formation of the upper flower in duplicated flower.3. The interaction patterns between most P. mume MADS-box proteins were similar to their orthologous genes with some genes diverged. The interaction patterns of PmFUL2 were similar to PmAP1, rather than PmFUL1. The complex formed by B class genes with other genes are combined by PmPI. The protein-protein interaction patterns among four E class genes were different, indicating the four E class genes control different flower organs. PmSEP2 and PmSEP3 can interact with A, B and C class genes; PmSEP3 can form complex with all four E-class genes.4. AP1/FUL subfamily genes and B class MADS-box genes were introduced into wild-type Arabidopsis by floral dipping method. PmAP1 can promote flowering and convert the inflorescence meristem to flower meristem. PmFULl can promote flowering and inhibit silique dehiscence. In addition to promote flowering and convert the inflorescence meristem to flower meristem, PmFUL2 control the development of flower organs. PmAP1 can promote flowering greatly while PmFULl weakly and PmFUL2 moderately. When introducing the three B class genes into wild-type Arabidopsis, no obvious phenotype change was observed.5. The model controlling floral organ development in P. mume was proposed according to the studies on the expression patterns and the protein-protein interaction profiles, the phenotype changes of wild-type Arabidopsis over-expressing P. mume MADS-box genes and comparative analysis of P. mume MADS-box genes with their orthologous genes. The flower meristem was specified by PmAPl, PmFULl, PmFUL2, PmSEP1 and PmSEP4; sepal by PmAP1, PmFUL2, PmSEP1 and PmSEP4; petal by PmFUL2, PmAP3, PmAP3-2, PmPI, PmSEP2 and PmSEP3; stamen by PmAP3, PmPI, PmAG, PmSEP2 and PmSEP3 and carpel by PmAG, PmSEP2 and PmSEP3.In this study, the whole MADS-box family genes were investigated first, then the functions of genes related to flower development were explored and the model that controlling floral organ development in P. mume was proposed. The results of this study will provide an effective and practical way to improve the ornamental traits of P. mume by molecular methods.