Cloning And Functioning Study Of APETATALI And AGAMOUS-LIKE 6 Homologs Of Magnolia Wufengensis

Belonging to Magnoliaceae,Magnolia wufengensis has variant floral shapes,color and tepal numbers within the species and is one of the ideal plants to study the flower origination and evolution.API is the critical MADS-box gene in ABCDE model to define the outer perianth identity,however recent reports proposed that AGL6 may have A class function in specifying the outer perianth floral organ characteristics for being highly expressed in tepals of basal angiosperm and basal dicots.The roles of APETATAL1 and AGAMOUS-LIKE 6 homologs were revealed in M.wufengensis tepal development by cloning their homologs and relatives in AP1/SEP/AGL6 super clade,analyzing their expression patterns,verifying their functions in transgenic Arabidopsis,revealing their protein-protein interaction patterns.The followings are the major results:1.Cloning and functioning study of API homologs of M.wufengensisMawuAPI was the only copy of the AP1-like that cloned from M.wufengensis,which had FUL motif and paleoAPl motif,and clustered with the other FUL-like homologs of basal angiosperms.The semi-quantative analysis showed that MawuAP1 was expressed both in leaves and floral organs;the quantative expression analysis showed that MawuAPI was highly expressed in bracts and carpels and its expression level gradually decreased from outer perianth to inner;the function analysis indicated that MawuAP1 could accelerate flowering and carpel development,but could not determinate the identity of outer perianth like its API in Arabidopsis.2.Cloning and functioning study of AGL6 homologs of M.wufengensis?1?Cloning and phylogenetic analysis of AGL6 homologsTwo AGL6 homologs of M.wufengensis were cloned,MawuAGL6 1 and MawuAGL6 2 with 81.0%identification of their coding region,81.1%of their amino acids and AGL6 motif I and AGL6 motif ? in their C domains.The analysis of the phylogenetic tree and expression pattern of AGL6 homologs in seeds plants proposed that MawuAGL6 1/2 were generated from the recent gene duplication event that happened in magnoliids.?2?Expression analysis of MawuAGL6₁/2The semi-quantitative analysis showed that MawuAGL6₁ was expressed in every whorl of M.wufengensis flower,while MawuAGL6 2 was not expressed in stamens;the quatitative expression analysis exhibited higher expression level of MawuAGL6₁/2 in tepals during the floral meristem formation and the rapid growth of the flora)buds before flowering;the expression of MawuAGL6 2 gradually decreased from outer-tepal to inner-tepal.These results suggested that MawuAGL6₂ may play the key role in defining the tepal identity.?3?Function analysis of MawuA GL6₁/2Arabidopsis tranformants of MawuAGL6₁/2 with wild type background showed early flowering;35S::MawuAGL6₁ caused early terminate flower and secondary flowers lacking outer perianth appeared between the first and second perianths of the terminate flower,which indicated that MawuAGL6₁ could promote flowering and determinate the flower meristem;35S::MawuAGL6₂ increased petal number of the transformed wild type Arabidopsis,which indicated that MawuAGL6 2 involved in petal identity specification;Arabidopsis transformed MawuAGL6₁/2 with apl-10 mutant background also showed early flowering;MawuAGL6₁ transformants showed leaf-like organs,petaloid stamens and secondary flowers appearing at the 1st and 2nd whorl of the terminate flower;MawuAGL6 2 recovered the petals and sepals of the mutants,and some also showed carpeloid sepals and stamen-like petals which indicated that MawuAGL6₂ could determine the identity of petals,sepals and carpels;both of MawuAGL6₁/2 had A class function.3.Protein-protein interaction analysis of MawuAP1,MawuAGL6₁/2 with B,C and E class MADS-box genes of M.wufengensis?1?Expression analysis of MADS-box genes of M.wufengensisQuantitative expression analysis of MawuAP1,MawuAGL6₁/2 and B,C,E class genes in 9 tepal M.wufengensis and its multi-tepal cultivar showed that only MawuAP1 was highly expressed in bracts;expression of MawuAGL6₂,MawuAP3₁/2 and MawuPI₁ in tepals were decreased from outer to inner tepals;MawuAGL6₂ were not expressed in stamens and the expression of MawuAGL6₁ was very low.These results suggested that MawuAGL6₁/2,MawuAP3₁/2 and MawuPI₁ may determine the tepal identity together.?2?Protein-protein interaction analysis of MADS-box genes of M wufengensisYeast two hybrid results showed that MawuAPl interacted with AGL6-like,API-like and SEP-like of M.wufengensis;MawuAGL6 1/2 interacted with AGL6-like,SEP-like homologs of M.wufengensis and Arabidopsis,furthermore,MawuAGL6₂ interacted with API homologs;all of them didn't showed any interations with B class genes;yeast three hybrid showed MawuAGL6 2 could interact with AtAP3 through AtSEP3 to specify the petal identity.