Molecular Mechanism Of AGL6 Homologous Genes Involving In Regulation Of Floral Development In Manglietia Patungensis Hu

Manglietia patungensis Hu,classified as a second grade protected wild endangered plant in China,is an everygreen ornamental tree producing white flowers with delightful fragrance and belongs to the family Magnoliaceae.M.patungensis owns lots of attractive traits suitable for garden landscaping and is also the northernmost distribution Manglietia species,which make it excellent model for investiagation flower evolution in Magnoliaceae.In angiosperms,AGL6?AGAMOUS-like6,AGL6?homologous genes play main roles in promoting flowering,specifying perianth and formation of axillary buds.The function and role of AGL6 homologous gene regulating the flower development of M.patungensis not only help to discover the molecular mechanism of tepals development and differentiation in M.patungensis,but also to understand the evolution of flower among different species in Magnoliaceae.Our results will also provide new gene resources for floral form improvement and germplasm innovation of the ornamental plants.The main results are as follows:?1?Cloning and Sequence analysis of AGL6 homologous gene in M patungensisThree MADS-box genes regulating floral development were isolated from flower buds of M patungensis by using homologous cloning method.Sequence alignment and molecular phylogeny analysis grouped them into the AGL6 lineage.The genes were therefore designated as Mapa AGL6A₁?Mapa AGL6A₂ and Mapa AGL6 B,respectively.Three sequences were also deposited in the Gen Bank under Accession Number KX926430?KX626431 and KX626432,respectively.Sequence analysis revealed that Mapa AGL6 A and Mapa AGL6 B in M.patungensis have arisen from a small scale gene duplication event.Both the genes contained a 729 bp ORF?Open Reading Frame?encoding the MIKCc-type MADS-box transcription factors with 242 amino acids length but 81.40% similarity.However,both proteins show mainly difference in the K-and C-domains.Moreover,the genomic sequence of the Mapa AGL6 A showed that Mapa AGL6A₁ and Mapa AGL6A₂ were derived from a consensus pre-m RNA.A57 bp exon skipping and 51 bp intron retention in the genomic sequence of Mapa AGL6 A produced Mapa AGL6A₂ transcprit,which encods a MADS-box transcription factor containing 240 amino acids.Moreover,the 57 bp exon skipping resulted in a truncated Mapa AGL6A₂ protein lacking 18 aa of the M-domain and 1aa of the I region,whereas the 51 bp intron retention resulted in a truncated Mapa AGL6A₂ protein with 17 aa insertion of the C terminal region,which resulted in the Tyr replaced by Cys in the AGL6 motif I.?2?Expression pattern analyses of Mapa AGL6A₁?Mapa AGL6A₂ and Mapa AGL6 B in M patungensisThe expression of MapaAGL6A₁?MapaAGL6A₂ and MpaAGL6 B were investigated among different floral organs and at flower buds at sequential developmental stages.The results showed that three genes were transcribed only in development tepals and were absent from the developing stamens,pistils and leaves.The relative expression values were changes in different petal whorls and in flowers with different tepals of M patungensis.In flower with 9 petals,three genes expressed mainly in the outer whorl sepaloid tepals at early stage of flower bud development,however,they expressed in the inner whorl petaloid tepals before blossom.Moreover,the expression patterns of three genes are same in different whorls of flower organs.In flower with 12 petals,the expression values of Mapa AGL6A₁ in inner petaloid tepals whorl showed significant high than that of it in two outer tepals whorls at early stage flower bud,while the expression of Mapa AGL6A₁ in three tepals whorls displayed no significant difference at late stage flower bud.However,the expression values of Mapa AGL6 B in sepaloid tepals whorl showed significant high than that of it in the inner petaloid tepals at late stage flower bud,while the expression of Mapa AGL6 B in three tepals whorls displayed no significant difference at early stage flower bud.Mapa AGL6A₁ and Mapa AGL6A₂ share a similar expression zones during the flower development,but the expression values of Mapa AGL6A₁ showed significant high than Mapa AGL6A₂ in flowers with 12 petals and 9 petals,respectively.?3?Function analyses of Mapa AGL6A₁?Mapa AGL6A₂ and Mapa AGL6 B in M patungensisWe produced transgenic Arabidopsis ap1-10?CS6230,Landsberg type?mutant lines to investigate the function of Mapa AGL6A₁?Mapa AGL6A₂ and Mapa AGL6 B,respectively.Phenotypes of transgenic lines were assayed in homozygous ap1-10 mutant backgrounds to evaluate whether ectopic expression of Mapa AGL6A₁?Mapa AGL6A₂ and Mapa AGL6 B could rescue the sepals and petals development,respectively.35S::Mapa AGL6A₁ transgenic plants produced flowers with sepaloid organ in whorl 1 and petaloid structure in whorl 2,and the bract-like structure and apical of bracts stigma-like structures in T0 and T1 generation were missed.It showed that Mapa AGL6A₁,as A function gene,mainly was involved in the development of perianth and restricted the expression of C function genes to the third and fourth whorls in transgenic Arabidopsis thaliana.However,it was found that only part of the plants produced sepal-like structure at the top of the filament in T0 of 35S::Mapa AGL6A₂ transgenic Arabidopsis In T1 generation,it appear a consistent stable genetic between with T0 of phenotypic characteristics,although few plants appeared the phenotypic of petal-like structure recovery but stigma-like structures of the bract apical did not disappear,It didn't inhibited function of class C gene in the outer two whorls;Phenotypic of 35S::Mapa AGL6 B transgenic Arabidopsis thaliana in T0 and T1 generation are relatively stable,which mainly appeared petal-like structure at the top of the filament and bract-like structure no change in two generation transgenic plant.It indicates that Mapa AGL6 B may be involved in formation of sepaloid organs,suggesting an obviously functional differentiation with the two splicing isoform of Mapa AGL6 A.