Cloning And Functional Analysis Of CpSGL2in Chimonanthus Praecox(L.)

Wintersweet [Chimonanthus praecox(L.) Link], belonging to Chimonanthus Lindl. of Calycanthaceae, with brilliant yellow petals, sweetperfume and beautiful appearance, is one kind of the ornamental bush in winter. The wintersweet is deeply enjoyed as cut flower and landscape plant in China. In present, there are many physiological and biochemistry researches on wintersweet flower,while few researches about the flowering molecular mechanisms of wintersweet MADS-box gene family, controling the floral organ development, is named after the initials of the four originally identified members:The yeast MINICHROMOSOME MAINTENANCE1(M.CM1) protein, the Arabidopsis thaliana AGAMOUS (AG), the Antirrhinum DEFICIENS (DEF) proteins and the human serum response factor (SRF). The MADS-box genes mainly involve in the process of regeneration, as well as control the vegetative growth, ovule development, root development and so on.We cloned an AGL2homologous sequence from the flower cDNA library of wintersweet. And then we analyzed the possible gene functions by bioinformatic analysis, transcriptional level in different tissues and floral development periods of wintersweet, and overexpression in Arabidopsis thaliana. The main results are as follows:1Molecular characteristics of CpAGL2Based on a cDNA library constructed from Chimonanthus praecox flowers, a new gene was cloned by randomly cloning and sequencing, named as CpAGL2. The CpAGL2gene has an open reading frame (ORF) of712bp, encoding a putative polypeptide of237amino acid residues. BLAST analysis showed that the gene sequence has a high homology with other plant. Bioinformatics analysis showed that the protein composed with45.57%α-helix,41.35%random coil and13.08%extended strand, and had10serine phosphorylation sites,3 threonine phosphorylation sites. Signal peptide and subcellular localization analysis showed that the protein may existed in nucleus possibly.2Transcriptional expression of CpAGL2It was showed that there was nearly no expression of CpAGL2in roots, stems and leaves, but higher in floral organs by Real-time fluorescence quantitative PCR. The pistils had highest expression level, and the outer petals had the second one. While, the expression levels of medial petals, inner petals, stamens were almost the same. There were little expression in the most early stage(Sprout period) of flower development. And then the transcript levels of the CpAGL2gene in the flower buds gradually increased in the early stages of flower development from flower-bud period to display-petal period. Then the expression the CpAGL2gene kept stable in initiating bloom period and bloom period. Finally, the transcript levels of the CpAGL2gene presented a peak at wither period. So it can be infered that the gene would has no deal with the early period of the bud differentiation, but may play some roles during the flower wither.3Expression analysis of CpAGL2in A. thalianaA plant over-expression vector pCAMBIA2301g-CpAGL2was constructed and transformed into A. thaliana successfully. Then, we selected the A. thaliana seeds until T2generation by medium containing kanamycin. GUS histochemical staining and RT-PCR analysis showed CpAGL2had expressed in A. thaliana, and we got6T2generation transgenic A. thaliana. There were no obvious phenotype change between transgenic and wild type of A. thaliana, and they also has the same flowering time. But the stamen numbers reduced in transgenic A. thaliana with a high over-expression of CpAGL2, so CpAGL2may have an effect on stamen development.