The development of the flower in plants is controlled by a series of genes that are influenced and regulated temporally and spatially by the internal and external factors. The classic ABC model of the flower development and regulation was proposed in 1991 (Coen and Meyerowitz, 1991; Weigel and Meyerowitz, 1994; Pelaz et al., 2000; Honma and Goto, 2001; Theissen and Saedler, 2001; Zhang et al., 2011), and now the ABCDE model was known and applied in the studies of the flower development in plant (Pelaz et al., 2000; Pelaz et al., 2001; Theissen and Saedler, 2001; Honma and Goto, 2001; Pinyopich et al., 2003; Jack, 2004; Ditta et al., 2004; Melzer and Theissen, 2009; Varkonyi-Gasic et al., 2011). In terms of the ABCDE model, the A+E genes specific referred to the sepalswhile the C+E genes specify the carpels; the A+B+E genes to the petals; the B+C+E genes to the stamensas well; Whereas the D gene individually specify the ovules. Exception of AP2, all of the homologous flower genes are members of the plant-specific MIKC MADS-box family (Ma and dePamphilis, 2000; Kaufmann et al., 2005; Adam et al., 2007; Xu et al., 2008; Varkonyi- Gasic et al., 2011). The MADS-box genes are vital in the formation of the floral meristem, development of the floral organs and florescence regulation in plants (Ng and Yanofsky et al., 2001; Becker and Theissen, 2003; Angenent et al., 2006; Guo, 2008; van Dijk and van Ham, 2010). In recent years, many studies have focused on the facets of the cloning, functional and comparative genomics and molecular evolution of the MADS-box genes.

Ornamental plants in the Guzmania genus, which belong to the Bromeliaceae family, are native to the American tropics and subtropics as well as are sold as superior-quality potted flowering plants. There is currently a lack of information on the Guzmania MADS-box gene. In this study, we atemped to clone the MADS-box gene from a Guzmania variety, Ostara, and analyze and express the genes in different tissues in Ostaras, In order to figure out the mechanisms of gene action in the flower development of Guzmania plant, which would be helpful to understand the floral meristem formation, development of floral organs, regulation of flowering time, and the molecular regulation and control of flower shape in Guzmania varieties.

1 Results
1.1 Cloning of the full-length cDNA sequence of GoMADS-box2
A 948 bp length cDNA sequence was assembled based on four EST sequences previously obtained from a full-length EST sequence library. To further validate the sequence, we completely sequenced ppfca 0_0002_G 08.z1.ab 1, one of the above 4 EST clonesby using a primer walking approach. BLASTp analysis showed that the 985 bp length sequence was identical to the above assembled 948 bp length sequence; therefore, we considered that both of sequences were the same sequence of the gene. The 985 bp cDNA sequence contains a 675 bp ORF (open reading frame) from the start of the 75^th base to the end of the 752^th base that encodes 225 amino acid residuals (Figure 1), which was named as GoMADS- box2 gene and deposited in GenBank with Accession number JN936044.

Figure 1 mRNA sequence and putative amino acid sequence of the GoMADS-box2 gene

1.2.2 Characteristic analysis of the deduced amino acid sequence of GoMADS-box2
ProtParam analysis indicated that the theoretical molecular weight and isoelectric point of the GoMADS-box2 gene be 26.3 kD and 9.36, respectively, and there were 28 negatively charged amino acids (Asp+Glu) as well as 36 positively charged amino acids (Arg+Lys). While secondary structure analysis with the SPOMA program revealed that the protein consisted of alpha helices (56.00%), random coils (24.44%), extended strands (13.78%) and beta turns (5.78%) (Combet et al., 2000). Conservative domain analysis by using the CDART program (NCBI) figured out that there were two typical conserved domains, the MADS and K-box domains in the gene (Figure 2) (Marchler-Bauer et al., 2011; Marchler- Bauer et al., 2009; Marchler-Bauer and Bryant 2004). The structures of MADS, I, K-box and C domains were located in AA2-60, AA61-70, AA71-170 and AA171-225, respectively (Figure 1), which should be the characteristics of plant-specific MIKC-type MADS-box proteins (Zheng et al., 2004; Folter et al., 2005; Immink et al., 2010; van Dijk et al., 2010).

Figure 2 Amino acid sequence of the conserved domain encoded by the GoMADS-box2 gene

1.2.3 Tertiary structure of GoMADS-box2 protein
Based on the 3D structure of the 1TQE P chain, a tertiary structure model was generated by the ESyPred 3D program (Figure 3), which determined that there was 19.1% homology between the 1TQE Pchain and the GoMADS-box2 protein (Lambert et al., 2002). The 2PSN structure, which was obtained by Han et al., in 2005 (Han et al., 2005), consisted of P, Q, R, S, X, and Y chains, and the P, Q, R, S chains were annotated as "transcription factor, MADS-box" by the InterPro program.

Figure 3 Thetertiary structure of the putative GoMADS-box2 protein based on the P chain of the 1TQE protein

1.2.4 Molecular phylogenetic tree of GoMADS-box2
To confirm the class that GoMADS-box2 gene belongs to, molecular evolution analysis was performed byusing the ABCDE model (Colombo et al., 1995; Pelaz et al., 2000; Xu et al., 2008). The evolutionary tree was generated by the MEGA 4.1 software with the neighbor-joining (NJ) method after a complete alignment was performed by using the Clustalx1.81 program. The results from the analysis were shown in Figure 4. Similar to AP3 (GI5805228, AAD51896.1) and PI (GI642128, D30807.1) of Arabidopsis thaliana, the GoMADS-box2 gene was assigned to B-class gene. Consequently, we thought that this gene might play an important role in the regulation and control of petal and stamen development.

Figure 4 The molecular phylogenetic tree of GoMADS-box2 based on the ABCDE model

Figure 5 Molecular phylogenetic tree of GoMADS-box2 based on Theissen's classification method (Theissen et al., 2000)

and GoGAPDH1), we determined the relative expression levels of GoMADS-box2 in various tissues (Figure 6), the results showed that GoMADS-box2 was expressed in all of the tested tissues, in which the floral expression level was the highest than that of other three tissues and 10 times more than that of the scape. According to t-test analysis between scape and leaf expression levels by using the PASW Statistics software (Ver. 18), the p value reached 0.307, indicating that was much more at 0.05 level, obviously, there was no significant difference between the two tissues. Whereas the relative expression level in the bract lowered to zero.

Figure 6 Expression levels of the GoMADS-box2 gene in various tissues

3.3.4 Real-time quantitative PCR analysis
The 25 µL volume real-time quantitative PCR reaction included 1 µL cDNA, 0.5 µL primer-F (20 pmol/µL), 0.5µl primer-R (20 pmol/µL), 12.5 µL 2×mix, 1 µL SybrGreen I (10×concentration) and 9.5 µL ddH₂O. The PCR procedures were listed in Table 1 performed in PRISM 7700 Sequence Detector ABI.

Table 1 Quantitative PCR procedures

3.3.5 Operation of apparatus
With the complete of the PCR preparation and procedures, the PCR amplification was performed on a 96-well plate in the Real-time Quantitative PCR System of ABI 7 700. To ensure a correlation coefficient closed to 1.00, the quality and concentrations of standard samples were adjusted.