Molecular Colning And Expression Analysis Of C-class MADS-box Genes From Lisianthus (Eustoma Grandiflorum)

Eustoma grandiflorum (Gentianaceae) is native to the central south of United States. Its fresh cut flower is very popular due to its strong adaptability, elegant flower type, long stem and various colour. There are cultivars with simple and double flower. In double flower, the development of four whorl organs are normal and the number of stamen is stable which are different from common phenomenon in other flower cultivars. In order to clarify the molecular mechanism of floral development, investigation was made on the conservation and divergence of expression pattern of floral organ identity MADS-box genes. The research used Eustoma grandiflorum yellow simple (No.49) and double flower (No.50) lines as experimental materials. AG-like subfamily MADS-box genes were isolated by RACE technology. Phylogenetic analysis and the specific spatial expression were investigated by which solid foundation was made for researching molecular mechanism of Eustoma grandiflorum floral development.(1) Gene 5'end sequences were indentified by RACE technology and the primers were designed by 3' end sequences. Three full length cDNA sequence of C-class MADS-box genes were isolated by RT-PCR. It makes foundation for analysis of gene evolution and expression.(2) To infer homology of these sequences to published C class genes, we first compared nucleotide and translated amino acid sequences with known genes for general similarities and presence of specific motifs. The amino acid sequences of EgPLE1, EgPLE2, and EgPLE3 showed high identities with that of AG and PLE. Multiple sequence alignments with other PLE-like proteins demonstrated that EgPLE1, EgPLE2 and EgPLE3 proteins have typical MIKC-type structural domain and the AG motifs, which are consensus sequence in the C domain of AG subfamily genes. Phylogenetic analysis showed these genes are most closely related to other core eudicots C-class MADS-box genes and supported the assignment of EgPLE1, EgPLE2 and EgPLE3 as AG (or PLE) orthologs. Genomic DNA was digested with restriction endonuclease and hybridized with specific probes of EgPLE1, EgPLE2 and EgPLE3 genes. The result indicated that EgPLE1 have two copies at least in the genome.(3) To investigate the spatial expression pattern of EgPLE1, EgPLE2 and EgPLE3 in simple and double flower lines of the Eustoma grandiflorum, gene-specific RT-PCR was performed with RNA that had been isolated from vegetative and floral organs. The result indicates that they express specially in stamen, carple and ovule but there is a difference that the expression quantity of EgPLE1 and EgPLE2 in simple flower's stamen decreases a little. The expression quantity of three genes in simple flower line are higher than that in double flower line and the difference between EgPLE1 and EgPLE3 are very evident. Prokaryotic expression was performed and we have got the EgPLE1 and EgPLE3 proteins. It makes foundation for protein structural analysis and transgene experiment.