Functional Study Of ADSPI And AktAP3-1 Of The Astragalus Membranaceus MADS-box Gene

Akebia trifoliata(Thunb.)Koidz,a medicinal vine of Lardizabalaceae(Akebia Decne.)which belongs to the basal eudicot speices,plays a transitional role in the evolution history of the angiosperms.Morphological studies on A.trifoliata flowers showed that A.trifoliata flowers possess bisexual flower structure in their early development stage,and the degeneration of the carpels of male flower and stamens of female flower in the late development stage leads the flowers to functional unisexual ones.It can be seen that A.trifoliata flowers develop in the way a unisexual flower of type I does,and yet the mechanism of molecular regulation during the process of transforming to unisexual flower from bisexual flower is remain unknown.The B class genes,belonging to the "ABCDE" model which accounted for the development of core eudicot flower,are the organ identity genes determining the development of petals and stamens and may participate in the sex differentiation of plants.In order to explore the molecular regulation mechanism of bisexual flower's transforming to unisexual flower.the preliminary studies of our research group indicated that the B class genes possess different temporal and spatial expression patterns in male and female flowers.Consequently,it is necessary to carry out a functional study on the B class genes of A.trifoliata.The B class genes belong to the MADS-box transcription factor family,and its encoded protein can form polymers by itself or by integrating with other transcription factors to regulate the transcription of the downstream target gene.Therefore,in order to further understand the regulating effect on flowers' sex differentiation and the interaction ways of encoded proteins of AktPI and AktAP3-1 genes which belong to the B class genes.In these studies,by ectopic transformation technique,the effects of AktPI and AktAP3-1 genes on the floral organ development on Arabidopsis thaliana.Meanwhile,the study explored the expression site and the interaction mode of AktPI and AktAP3-1 proteins in the plant cells by subcellular localization technique and bimolecular fluorescence complementary technique.The function of AktPI and AktAP3-1 genes were preliminarily revealed in the research results,which was of great significance to understand the effect of them on the molecular regulation mechanism of the A.trifoliata development and to further explore the molecular mechanism of bisexual flower's transforming to unisexual flower of the A.trifoliata.The results and conclusions of the study were as follows:1.Study on the ectopic expression of AktPI and AktAP3-1 genes in Arabidopsis thaliana.AktPI and AktAP3-1 transgenic plants showed an increase in stamens.Combined with the preliminary research results of this kind of gene proposed by the research group adopting real-time quantitative method,it can be deduced that the AktPI and AktAP3-1 genes can promote the development of stamens,and inhibit the development of pistil,and may be involved in the formation of unisexual flower of A.trifoliate.In addition,AktAP3-1 and AktPI transgenic plants showed early flowering,AktAP3-1 transgenic plants showed curly cauline leaves and terminal flowers.Meanwhile,in the first round of the terminal flower there were three extra incomplete flowers lacking of parts of the floral organ,forming a normal developed pods and a little dysplastic pods,which indicated that AktAP3-1 also may be involved in the transformation from inflorescence meristem to flower meristem and is related to the development of pods.2.Study on the subcellular localization of AktPI and AktAP3-1 genes encoded proteins.p1302-AktPI-GFP and p1302-AktAP3-1-GFP fusion proteins expression vectors were constructed,respectively transforming the onion epidermal cells and tobacco cells.The results showed that p1302-AktPI-GFP and pl302-AktAP3-1-GFP fusion proteins were located to the cell membrane and cytoplasm of tobacco cells and onion cells,which indicated that AktPI and AktAP3-1 proteins do not possess nuclear localization ability,and they may need to be integrated with other transcription factors to form a polymer,taking part in the regulation of A.trifoliate flower organs development.3.Study on the interaction between AktPI and AktAP3-1 genes encoded proteins.To construct the transient expression vectors of pYNE-AktPI,pYNE-AktAP3-1,pYCE-AktPI and pYCE-AktAP3-1 to transform the tobacco cells and observe YFP fluorescent protein renaturation.Results showed that the AktPI and AktAP3-1 proteins can form a heterodimer,and also can respectively form a homodimer.And the location of yellow fluorescence in the cytoplasm of tobacco cells indicated that the homodimer or heterodimer formed by AktPI and AktAP3-1 proteins can be located in the cytoplasm of tobacco cells.It can be deduced that with the evolution of angiosperms and the increase of paralog genes,B-type protein tends to form heterodimer and its gene network of regulating the development of flower organ is becoming increasingly complicated.