The Function Study Of AktFL1 And AktAG1 Of Sanye Mutong MADS-box Family

Akebia trifoliata(Thunb.)Koidz is a medicinal vine of Lardizabalaceae(Akebia Decne.),belonging to the group of the basal eudicot speices.It has an important position in the evolution of angiosperms.For the morphological study of A.trifoliate flowers,it was found that:at the beginning of development,they were bisexual flowers.The stamens of the female flowers and the carpels of the male flowers were degenerated in the late development stage respectively,thus forming the functional unisexual flowers.The development pattern of A.trifoliata flower was known as unisexual flower of the type I.However,it is still unclear that how bisexual flower transformed to unisexual flower.Studies have shown that sex-determining genes,epigenetic modifications and phytohormones are the influencing factors for the sexual differentiation of unisexual flowers.The genes that modulate the sexual differentiation of parthenogenetic flowers belong to the MADS-box gene family.According to the flower development "ABCDE"model,the C class genes are not only the organ-specific genes that determine the development of stamens and carpels,but also it may be a key factor in regulating the sexual differentiation of plants.The A class genes are not only organ characteristic genes that determines the development of sepals and petals,but also have functionally antagonistic relationship with the C class genes showed by studies in model organism Arabidopsis.In order to explore what the molecular mechanism of the unisexual flower formation of A.trifoliate flower is and explore the relationship between class A and C genes in flower development,it is necessary to classify the function of A class genes and C class genes.Therefore,in order to understand the mode of action of A class and C class genes and their encoded proteins in A.trifoliata,AktFLl gene in A class gene and AktAGl gene in C class gene were studied.The preliminary analysis of them was performed by semiquantitative RT-PCR.The effects of them on the floral organ development of Arabidopsis thaliana were further studied by using ectopic transformation technique,AktFL1 and AktAGl genes were also explored using subcellular localization technique and bimolecular fluorescence complementary technique,respectively.The function of AktFL1 gene and AktAGl gene were preliminarily revealed in the results.It is of great significance to further explore the molecular mechanism of the unisexual flower formation of the A.trifoliate.The results and conclusions of this study are as follows:1.Semi-quantitative RT-PCR analysis of AktFL1 gene and AktAG1 gene.According to the changes in the height ratio of the stamens and carpels of A.trifoliata,the development process was divided into four periods.The total RNA of male and female flowers from the 1st to 4th stages of the flower development stage was used as the amplification template.18S rRNA was selected as the reference gene.The results showed that the expression level of AktFL1 gene increased first and then decreased in female flowers,but decreased first and then increased in male flowers.The AktAGl gene showed a tendency of increasing.first and then decreasing in the male and female flowers.In the 2nd and 3rd stages,the female flower expression was slightly higher than that of the male flower,but it was basically the same in the 4th period.It was concluded that the AktFL1 gene and the AktAGl gene exhibited no antagonism in their expression.2.Overexpression of AktFL1 gene and AktAG1 gene respectively in Arabidopsis thaliana.The 35S::AktFL1 and 35S::AktAG1 overexpression vectors were constructed and transformed to Arabidopsis thaliana.The stamens increased in AktAG1 transgenic plants.It was concluded that the AktAG1 gene promotes stamen development and participates in the regulation of unisexual flower formation.The color of sepals which belong to AktFL1 transgenic plants appeared to lighter.It was speculated that the AktFL1 gene is involved in the regulation of the sepal development.In addition,both AktFL1 and AktAG1 transgenic plants showed early flowering and both of them had 2-3 apical flowers,indicating that both AktFL1 gene and AktAG1 gene can promote the transition from vegetative growth to reproductive growth in Arabidopsis thaliana.Therefore,the functional antagonism of AktFL1 and AktAG1 genes has not been found,and further verification is needed.3.Subcellular localization of proteins encoded by AktFL1 gene and AktAG1 gene respectively.The p1302-AktFL1-GFP and p1302-AktAGl-GFP fusion expression vectors were constructed and transformed into the tobacco cells and the onion epidermal cells,respectively.The results showed that both p1302-AktFL1-GFP and p1302-AktAGl-GFP were localized in the cytoplasm of tobacco cells and onion cells.Indicating that both AktFL1 and AktAGl proteins have no nuclear localization ability.It is speculated that they need to be combined with other transcription factors to form the complex input nucleus,thereby regulating the development of A.trifoliate flower organs.4.The interaction between AktFL1 and AktAG1 genes encoding proteins.The transient expression vectors pYNE-AktFL1,pYNE-AktAG 1,pYCE-AktFL 1 and pYCE-AktAG1 were constructed and transformed into the tobacco cells to observe the renaturation of YFP fluorescent protein.The experimental results show that AktFL1 and AktAGl proteins can both form homodimers and heterodimers,respectively.Moreover,referencing the location of YFP fluorescence in tobacco cells,it can be concluded that the homologous or heterodimer formed by AktFL1 and AktAG1 protein is localized in cytoplasm of the tobacco cells.It shows that with the continuous evolution of angiosperms,A-type proteins and C-type proteins have evolved a variety of interaction patterns in different plants.In this study,semi-quantitative RT-PCR technology was used to preliminarily explore the expression patterns of these two genes,and it was concluded that there was no mutual antagonism between AktFL1 and AktAGl genes;further studies were conducted using Arabidopsis ectopic transformation techniques to study the two approaches.The regulation function of flower organ development in A.thaliana resulted of that AktAGl gene promoted the development of stamens and participated in the regulation of the formation of unisexual flowers of A.trifoliata.It has not been concluded that AktFL1 and AktAG1 have mutual antagonistic effects on function,and further confirmation is needed;Using subcellular localization techniques,the proteins encoded by these two genes are localized in the cytoplasm;using two-molecule fluorescence complementary techniques,the proteins encoded by the two genes can both form homodimer and heterodimer respectively.In vivo,the homo-or heterodimer formed is localized in the tobacco cytoplasm.The results of these studies will provide a theoretical basis for exploring the molecular mechanism of bisexual flower flowers forming parthenogenetic flowers and lay a foundation for the establishment of a gene regulatory network for the development of A.trifolia.