Cloning And Expression Analysis Of AktAG1 And AktSEP3 Genes Of Akebia Trifoliate

Akebia trifoliata(Thunb.)Koidz is a deciduous woody vine,which belongs to Lardizabalaceae.It is distributed mainly over Hebei.Shanxi.Shandong.Henan?southern Shaanxi?eastern Gansu and the Yangtze River Basin."The Pharmacopoeia of the people's Republic of China" of 2010 edition rule dry stem and nearly ripe fruit of Akebia trifoliate as Mutong and Yuzhizi.It is an important medicinal plant.The flowers of Akebia trifoliate are monoecious,inflorescence with diversity,It includes perfect flower inflorescences,male inflorescence and female inflorescence,the morphology of female and male flowers is bisexual during the initiation and early development of flowers,have six stamens primordia and 3-9 carpel primordia,The late stage of floral development,the flowers become unisexual,the stamens are degenerated in female flowers and the carpels become rudimentary in male flowers.The present study shows that the flower development is influenced by the genes,chromosomes,environment and so on,and it is different according to the plant type,all the genes in the ABCDE model,except AP2,belong to the MADS-box gene family,and the AG gene is the C Class gene,They have an important regulating effect in the development process of pistil and stamen,a large number of studies shown that,the AG gene have conserved function in determining reproductive organ identities in the model plant?the main crops and the important economic flowers.The SEP3 gene is required for the development of each floral organ.In order to explain the functional and molecular mechanism of C and E class genes in the development process of pistil and stamen,and to study whether C and E class genes has a regulation role during the Akebia trifoliate flowers from bisexual to unisexual.In this study,two MADS-box gene were cloned from the different developmental stages of male and female flower organs by the method of homologous cloning,RACE and other technical.Phylogenetic relationship and expression patterns analysis understand the AktAG and AktSEP3 gene expression in Akebia trifoliate sex determination.The main experimental results:(1)observing the morphologic of flowers during the development of unisexual flower in Akebia trifoliate,The flowers of Akebia trifoliate are monoecious.Using the microscope observation,aceerding to the highproportion of carpd and stamen,floral development were segmented into four periods.stages 1:male and female inflorescence not obvious,stages 2:base male flower carpel and stamen height ratio is greater than 1/2 and less than 1,female flower carpel and stamen height ratio is greater than 1 and less than 2,stages 3:stamens of male flowers was significantly higher than that of the carpel,stamen and carpel height is less than 1/2,the carpel of female flowers was obvious higher than that of stamens,carpels and stamens height ratio is greater than 2,stages 4:carpels in the male has been reduced to very small or absent,female flower stamens degenerate and disappear.(2)Extracting total RNA from the flowers of Akebia trifoliate,this study selected four kinds of plant RNA extraction methods,including CTAB-Licl method,SDS-acid phenol method,Tris-boric acid method and RNA extraction kit.The results showed that the total RNA isolated with CTAB-Licl method and plant RNA extraction kit had high integrity,high yield and good stability.(3)AktAGl and AktSEP3 gene cloning from Akebia trifoliate,AktAG1 and AktSEP3 genes has been isolated from Akebia trifoliate the way of homologous cloning combined with RACE technique.Translate their amino acid sequence,we can see that these two genes have a typical M,K,I,C domains belonged to MIKC-type MADS-box genes.The full-length of AktAGl cDNA is 1009 bp,containing an open reading frame of 738bp encoding a 245 amino acids,belonged to AG-like gene subfamily.The full length of AktSEP3 cDNA is 1002bp,containing an open reading frame of 729bp encoding a 242 amino acids,belonged to AGL9 gene subfamily.Sequence homology and phylogenetic analysis revealed that these two genes and basal angiosperm have recently relationship,indicating that the species closer genetic relationship,the higher sequence homology.(4)The expression of Akt AG l and AktSEP3 gene from Akebia trifoliate,semi-quantitative RT-PCR analysis indicated that AktAGl and AktSEP3 genes were expressed in Akebia trifoliate floral organs,AktAGl low expression in the calyx,it was highly expresses in carpel and stamens,it was not detected expression in vegetative organs.in the female expression at the stages of flower development is increase and then decrease,in the male expression unchanged.AktSEP3 expression remained unchanged in different floral organs and different stages of flower development.