The Molecular Mechanism Of Key Genes PhAP3 And PhCYC Involved In Bisymmtry Flower Development In Phalaenopsis Hybrid

Orchidaceae are widely distributed all over the world which have extremely excellent ornamental and economic values.Because of its highly specialized lip and staman,it is an ideal material for studying the development and evolution of angioserm floral organs.As the most commercialiazed orchid,Phalaenopsis has a short growth cycle and mature flowering induction technology.Therefore,in-depth studies on the molecular mechanism of its flower bisymmtry development could provide a basis for the improvement of orchid characteristics and the breeding of new orchid varieties with independent intellectual property rights.The APETALA3(AP3)and CYCLOIDEA(CYC)genes in model plants have significant effects on the development of floral organs,but their functions and regulatory relationships in Phalaenopsis are still unclear.Our previous study found that there are three CYC-like homologous genes in Phalaenopsis equestris via searching the Orchid Base 3.0,and successfully cloned them and obtained corresponding transgenic plants of Torenia fournieri,which are speculated to be involved in the development Phalaenopsis.Based on these results,we used Phalaenopsis hybrid ‘Little Gem Stripes’ as the material in this study and cloned two AP3 homologous genes and the function of genes were analyzed through real-time q RT-PCR and transgenic technique.Then we used the yeast hybrid,dual-luciferase assay and transient expression methods to explore the molecular mechanism of PhAP3 s and PhCYCs genes regulating the floral organ development of Phalaenopsis.The main results are as follows:1.Two AP3 homologous genes were cloned from Phalaenopsis hybrid ‘Little Gem Stripes’,which named PhAP3-1 and PhAP3-2.Sequence alignment and phylogenetic analysis revealed that two PhAP3 homologous genes contained conserved MADS and K domains,PI-derived motifs and paleo AP3 motifs,which belonged to the paleo AP3 clade Ⅰand Ⅲ of orchids respectively.Analysis of expression patterns using the wild-type flower buds and mutant petal-like stamans of Phalaenopsis as materials,the expression level of PhAP3-1 in lateral petals is higher than that of PhAP3-2,while PhAP3-1 is significantly lower than PhAP3-2 in the lip.The expression patterns of PhAP3-1 and PhAP3-2 in the mutant indicate that PhAP3-1 played an important role in the formation of petal-like stamen.The transcriptional activation activity analysis and the yeast two-hybrid show that PhAP3-1and PhAP3-2 didn’t have transcriptional auto-activation ability.Both genes could form heterodimers with PhPI,but they could not form homodimers by themselves.2.The overexpression vectors of PhAP3-1,PhAP3-2 were constructed and transferred into Arabidopsis by Agrobacterium-mediated method for phenotypic investigation.The results showed that the floral symmetry of the PhAP3-1 and PhAP3-2 transgenic lines displayed significant changes compared with the wild-type plants,the number of stamens decreased and degenerated in 35S::PhAP3-2 lines.In addition,the transgenic plants of PhAP3-1 and PhAP3-2 showed that the phenomoenon of promoting flowering under long-day condition.The real-time q RT-PCR demonstrated that the expression level of flowering promoting genes AP1,FT,FUL increased significantly,while the expression level of flowering inhibiting gene TFL1 decresed.3.Through the expression pattern analysis,transcriptional activation analysis and yeast two-hybrid of three PhCYC homologous genes,we found that the expression of PhCYC1 in the lip and lateral petals was significantly higher than that of PhCYC2 and PhCYC3,none of them had the ability to activate transcription.Meanwhile,there were no protein interactions between each other.Additionally,three PhCYCs partial promoter sequences were cloned,all of which contained CAr G-box cis-acting elements.The interactions between PhAP3 s and PhCYCs were explored by yeast one-hybrid,dual-luciferase assay and Phalaenopsis transient transformation assay.PhAP3-1 and PhAP3-2 were proven to bind with the promoter of PhCYC1,and the predicted promoter binding sites were-634 bp at the upstream region of the PhCYC1 initiation codon(ATG).PhAP3-1 and PhAP3-2 proteins interacted with PhCYC1 promoters as a transcription activator,which activate the expression of PhCYC1.The result was also confirmed by Phalaenopsis transient transformation,as transient transformation of PhAP3-1 and PhAP3-2 resulted in increased expressions of PhCYC1.These results indicated that PhCYC1 may be involved in the development of the lip regulated by PhAP3-2.