Molecular Colning And Expression Analysis Of Floral Organ Identity MADS-box Genes From Lisianthus (Eustoma Grandiflorum)

Eustoma grandiflorum Grise.is a gentian native to the central and southern United States. Due to its large flowers,long stems,a wide range of flower colors and extended vase life,its cut flowers become increasingly popular.There are cultivars with simple and double flowers. In double flowers,the developments of four whorl organs are normal and the number of stamens is stable.In order to elucidate the molecular mechanism of floral development, investeigation was made on the conservation and divergence of expression patterns of floral organ identity MADS-box genes.Several MADS-box genes,including DEF/AP3 subfamily(B class),GLO/PI subfamily(B class),PLE/AG subfamily(C class),and SEP3/AGL9 subfamily (E class),were isolated from Eustoma grandiflorum,yellow simple(No.49) and double flowers(No.50) lines,by SSH(suppression subtractive hybridization) and 3'-RACE(Rapid amplification of cDNA ends).Sequence analyses,phylogeny reconstruction and RT-PCR expression analyses were performed.The gene structure and expression obtained in this investigation can provide important information regarding the floral development of Eustoma grandiflorum.1.Primary screening of floral organ identity genes during floral development with suppression subtractive hybridization in Eustoma grandiflorumThe purpose of this study was to get floral identity genes from Eustoma grandiflorum using SSH.A subtractive cDNA library,which was enriched in genes related to flower organ characteristics,was constructed.The cDNA prepared from sepal,petal,androecium,and gynoecium at different developmental stages was used as the tester and the cDNA from stem and leaf organ as the driver.From the library,1610 high quality sequences(longer than 200 bp, 696 bp at the longest and 341 bp in average) were isolated.After clustering,these unigenes were contigged by SeqClust 2.1 software,and then we got 667 contig sequences.By running blastn program of NCBI,330 contig sequences were found to have homologous genes with the network database,and 337 ESTs(expressed sequence tags) without remarkable homology. Among 330 ESTs with homology,there were 310 ESTs with known function and 20 ESTs with unknown function.The results of GenBank search indicated that they showed a high degree of identity in nucleotide sequence with DEF gene from Antirrhinum majus,AP3 gene from Ilex aquifolium,MADS3 gene from Vitis vinifera,FBP3 gene from Petunia hybrida,and GGM6 gene Gnetum gnemon,respectively.These results showed that the subtractive cDNA library was constructed successfully and that the foundation was established for cloning the floral organ identity genes. 2.Isolation and phylogenetic analysis of MADS-box genes from Eustoma grandiflorumPlant MADS-box genes encode a family of highly conserved transcription factors that are involved in many different developmental processes including flower development.In order to elucidate the molecular mechanisms of floral development in a dicotyledonous species, lisianthus,total RNA prepared fron flower buds and mature flower organs,cDNA of five flower-specific MADS-box genes were isolated from this plant by the method of 3'-RACE using degenerate primers designed according to the conserved region of MADS-box protein family taken from flower bud.Sequence and phylogenetic analysis indicated that they had a high degree of identity in the predicted protein sequence with DEF gene from Antirrhinum majus,FBP3,FBP6,and FBP7 genes from Petunia,and SEP3gene from Arabidopsis thaliana,respectively.Therefore,these five genes were termed EgDEF1(DEF/GLO subfamily, B class),EgGLO1(DEF/GLO subfamily,B class),EgPLE1(AG-like subfamily,C class), EgMADS1(AGL11-like subfamily,D class),and EgSEP3-1(SEP-like subfamily,E class), respectively.Alignment of the predicted amino acid sequences from these genes,along with previously published subfamily members,demeonstrated that these genes comprise four regions of the typical MIKC-type MADS-box proteins:the MADS domain,intervening(I) domain and keratin-like(K) domain,and the C-terminal domain.Alignment of C-terminal regions of the predicted protein sequences of these genes with those of their subfamily genes revealed that each gene bears the highly conserved motifs.3.The expression of the MADS-box genes from Eustoma grandiflorumTo investigate the expression patterns of the floral organ identify MADS-box genes in flower of Eustoma grandiflorum,gene-specific RT-PCR was performed with RNA that was isolated from vegetative and floral organs.This analysis revealed that both class B-function genes EgDEF1 and EgGLO1 were strongly expressed in petals and stamens.The EgDEF1 expression was strongly detected in sepals and ovules,but the EgGLO1 expression in sepals was weakly detected with no detection in ovules.C-function gene EgPLE1 was expressed specifically in stamens,carpels,and ovules,but the expression in stamens less abundant.D-function genes EgMADS1 was expressed specifically in carpels and ovules.E-function genes EgSEP3-1 expression was strongly detected in four flower whorls.Theses experiments revealed that five MADS-box genes were expressed specifically in floral organs,while no signal was detected in vegetative organs such as root,stem,and leaf.