Cloning And Functional Analysis Of PaAG2 And PaAG3 Genes In Platanus Acerifolia

London plane(Platanus acerifolia)is a woody plant of Platanaceae family,which belongs to the relatively primitive basic dicotyledonous group in the evolutionary position.It is a hybrid of American sycamore(P.occidentalis)and Oriental plane(P.orientalis)which has a beautiful shape and good growth shape.It has a very important position in garden applications and has the reputation,which is also an excellent landscaping plant praised as ‘the king of street trees’ and is widely planted in roadside and courtyard for shading.However,countless flowers and fruits with pollens and seed hairs in spring from the adult trees has reduced its application value.Therefore,in order to solve this problem,the cultivation of the fruitless London plane has a high practical value.The purpose of this study is to study the molecular mechanism of flower development in two species of Platanus acerifolia from the molecular level,thereby regulating its flowering to achieve infertility.The AGAMOUS gene is a key gene in flower development model affecting stamen and carpel development.Therefore,it is clear that the expression pattern and functional differentiation of AGAMOUS homologous genes in Platanus acerifolia have a significance for understanding the regulation of Platanus acerifolia and cultivating sterile plants.The main results are as follows:1.The two homologous genes of AGAMOUS gene in Platanus acerifolia were cloned,named PaAG2 and PaAG3.The full length of CDS was 678 bp,and the predicted encoding acids was 225 bp.The amino acid sequence alignment showed that PaAG2,PaAG3 and the reported PaAG1 all possessed the complete M,I,K,and C domains of the MADS-box gene and found the unique AG I and AG II motifs of AG family genes in the relatively least conserved C-terminal region.2.There are similarities and differences in the expression patterns of the AGAMOUS gene homologous genes PaAG1,PaAG2 and PaAG3 in Platanus acerifolia.In general,the peak of expression level is concentrated in the fruit development stage,the expression level in female inflorescence is second,and the expression level of male inflorescence is the lowest.PaAG2 is higher than the expression of the other two AG homologs during the development of the whole fruit development stage,and is more specifically concentrated in the fruit.PaAG1 is mainly expressed specifically in the reproductive organs and is not expressed in vegetative tissues.PaAG3 also expressed in the roots and mature leaves of some vegetative tissues.3.Two over-expression vectors 35S: PaAG2 and 35S: PaAG3 were established,and the over-expression tobacco and Arabidopsis thaliana were functionally verified.The sepals of the transgenic tobacco phenotypic lines overexpressing PaAG3 are clearly carpelled,forming an elongated stigma at the apex,and forming a fusion that envelops all the inner round flower organs,making the petals,stamens and carpel all died.The petal changes to the stamen,and the flower tube transforms into an independent filament.The anther-like structure appears at the top of the petal,indicating that PaAG3 has the role of regulating the third and fourth round flower organs.Among the over-expressed Arabidopsis thaliana,35S:PaAG2 and 35S:PaAG3 can affect the vegetative growth and reproductive growth of plants and 35S:PaAG3 also has the function of promoting early flowering.It is indicated that there is functional redundancy between AG homologs in Platanus acerifolia in controlling floral organ development.4.Yeast two-hybrid assay PaAG2 has strong interaction with the B gene PlacPI2 a and the E gene PlacSEP1.PaAG3 interacts with the B gene PlacPI2 a,PlacAP3 and the E gene PlacSEP1 and PlacSEP3,indicating that the PaAG2 and PaAG3 genes in the Platanus acerifolia play the most conservative C-class gene function.