Functional Analysis Of Flowering-related LFAFY、UFO And MADS-box Genes In Metasequoia Glyptostroboides

Metasequoia glyplostroboides Hu&Cheng is a national first-class protected plant native to China,known as the "living fossil".Metasequoia has the characteristics of long juvenile phase,which limits the process of afforestation.Based on the first EST database of Metasequoia constructed and the expression profile of the male and female flower bud differentiation period in the early stage of the laboratory,we cloned the differentially expressed genes in the flower bud differentiation and flower organ formation process,including two LFY(LEAFY)families members,3 UFO(UNUSUAL FLORAL ORGANS)gene family members the and 14 members of the MADS-hox family;analised the interaction between LFY and UFO proteins by yeast two-hybrid technique;overexpressed the A4gLFY and MgNLY genes in Arabidopsis to study gene function.The expression pattern of MADS-box gene was analyzed by qRT-PCR technology,and the interaction mode and characteristics of MADS-box protein were verified by yeast two-hybrid technique.The main results are as follows:(1)The microstrobilus of Metasequoia are usually born at the top of the branches,and the open position facilitates the spread of pollen by means of wind.The mature sporangioles is longitudinally cracked,and the three sporangioles are successively cracked,which can prolong the loose powder time and avoid the adverse effects on pollen.(2)In order to study the gene expression of different metasequoia and hormone treated Metasequoia,the internal reference gene stably expressed qRT-PCR was screened,and 14 candidates were selected by ΔCt,Bestkeeper,NormFinder,geNorm,RankAggreg and GrayNorm.The expression stability of the reference gene in tissues and hormone treatment was analyzed.On the basis of the ranking of gene expression stability,Analysis using the GrayNorm algorithm showed that ACT2(Actin 2),HIS(histone superfamily protein H3)and TATA(TATA binding protein)were stably expressed in different tissues.ACT2,EF1α(elongation factor-1 alpha)and HIS were optimal for leaves treated with the flowering induction hormone solution,while Cpn60β(60-kDa chaperonin β-subunit),GAPDH(glyceraldehyde-3-phosphate dehydrogenase)and HIS were the best reference genes for treated buds.EF1α,HIS and TATA were useful reference genes for accurate normalization in abscisic acid-response signaling.Our results emphasize the importance of validating reference genes for qRT-PCR analysis in Metasequoia.(3)Through the phenotypic observation of 35S::MgLFY and 35S::MgNLY transgenic Arabidopsis lines,it was found that 35S::MgLFY and 35S::MgNLY transgenic Arabidopsis lines flowered earlier than wild-typel,MgLFY,MgNLY can induce Arabidopsis flowering in advance,thus shortening the flowering cycle.(4)By means of yeast two-hybrid technique,it was found that there are interactions between MgLFY and MgNLY with their own proteins,and homologous complexes can be performed.There is also interaction between MgLFY and MgNLY proteins,which can form heterologous complexes.The MgLFY and MgNLY genes can interact with the UFO 1 and UF02 genes,respectively.Analysis of the mode of action of the B-type gene members of MADS-box revealed that there is no interaction between the three proteins of MgAP3-2,MgAP3-3 and MgPI,There is no interaction between the three proteins MgAP3-2,MgAP3-3 and MgPI,but there is an interaction between MgAP3-2 and MgAP3-3 with MgPI protein,respectively,and a heterologous complex can be formed.In summary,the LFAFY gene of Metasequoia can interact with UFO.It is speculated that the UFO gene in Metasequoia may act as a transcriptional cofactor to regulate the activity of LFY gene and participate in the regulation of floral meristem development and flower organ identity.The interaction pattern of the B gene is similar to that of other species.This way of regulating the formation of floral organs by forming a heterologous complex is conservative in plants.The results provide an important theoretical basis for studying flower bud differentiation and flower organ formation,and also provide guidance for exploring the related research of gymnosperm flower development.