| Tree peony (Paeonia suffruticosa Andr.) is the top one of ten famous flowers in China and is a symbol of beauty, happiness, good fortune and prosperity. It is deeply loved by people all over the word. Normally, tree peony blooms in May or April every year. Although tree peony can bloom during the Spring Festival by the flower forcing techniques, it still cannot satisfy people’s desire of enjoying flowers because the florescence of tree peony is only seven days or so. Both ornamental quality of tree peony and economic benefit for planting tree peony are affected by its short florescence, which restricts the development of tree peony industry. Therefore, it is of great urgency and significance to delay the senescence of tree peony and prolong its florescence.Tree Peony’Lu He Hong’was used as plant materials in the present study. By observing the changes of morphological and microscopic structures of tree peony senescence under natural conditions, we confirmed the stage of flower senescence. Two cDNA libraries associated with senescence were constructed using the technology of suppression subtractive hybridization (SSH). The full length of cDNA for three genes was obtained by RACE amplification and the bioinformatics analyses were conducted. The overexpression vector pBl121-PsMADS4 was constructed and wild-type Arabidopsis was transformed by Floral-dip. Then the phenotype of PsMADS4 transgenic Arabidopsis was observed. The main results are as follows:1. Based on the observation of the morphology of flower blooming under natural conditions and the paraffin section of the petal, we found the trend was consistent between the changing of the petal’s structure and its morphology at different stages of its development. The aging period of tree peony was confirmed ultimately.2. Two SSH cDNA libraries associated with flower senescence were constructed using blooming petals and senescent petals as tester and driver respectively. A total of 211 significant Expressed Sequence Tags (ESTs) were obtained. According to the results of the Kyoto Encyclopedia of Gens and Genomes databases (KEGG),48 ESTs were functionally annotated. After the orthologous clustering analysis of the 211 ESTs by using COG Corpus, we found that 44 EST were classified into 14 functional categories as follows: (1) General function prediction only (2) Carbohydrate transport and metabolism (3) Posttranslational modification, protein turnover, chaperones (4) Translation, ribosomal structure and biogenesis (5) Lipid transport and metabolism (6) Inorganic ion transport and metabolism (7) Cytoskeleton (8) Transcription (9) Cell wall/membrane/envelope biogenesis (10) Intracellular trafficking, secretion and vesicular transport (11) Defense mechanisms (12) Amino acid transport and metabolism (13) Secondary metabolites biosynthesis, transport and catabolism (14) Replication, recombination and repair. In order to further analyze the transcription levels of differentially expressed genes at different stage of flower senescence, the qRT-PCR analysis on the 23 ESTs which were selected randomly from the 211 EST were performed. It shows that the results of qRT-PCR are consistent with the results of SSH libraries.3. Based on the EST of Long-chain Acyl-coenzyme A synthetase (LACS) from SSH library, the full length of cDNA was obtained by RACE amplification and was named as PsLACS. The length of PsLACS cDNA is 2377 bp including 1830 bp encoding frame, which encodes 609 amino acids. Its molecular weight is predicted as 67.94 kilodaltons, and pI is 6.191. The PsLACS has 30 possible phosphorylation sites, which might be associated with the regulation of enzyme activity. The predication results of the secondary structures showed that PsLACS mainly contained a-helix and random coil. Alignment analysis showed that PsLACS has the highest homology with Vitis vinifera LACS, followed by Citrus sinensis LACS and Ricinus communis LACS, which is basically consistent with the results gotten from the phylogenetic analysis.4. Plasma membrane intrinsic proteins (PIPs), as one kind of Aquaporins, play a key role in transmembrane water transport. Based on the EST of PIPs from SSH library, the full length of cDNA was obtained by RACE amplification and was named as PsPIP2. The length of PsPIP2 cDNA is 1174 bp including 846 bp encoding frame, which encodes 281 amino acids. Its molecular weight is predicted as 29.79 kilodaltons, and its pI is 9.04. The predication of phosphorylation sites shows that the amino acid sequence of the PsPIP2 contains 8 possible phosphorylation sites, which might be associated with the openness and closure of channel in PsPIP2. The predication results of the secondary structures showed that PsPIP2 mainly contained random coil and a-helix. Alignment analysis showed that PsPIP2 had the highest homology with Manihot esculent a PIP2.5. As an EST extracted from SSH library, the transcriptional level of PsMADS4 is significantly increased in the blooming flowers. The full length of cDNA was obtained by RACE amplification and was named as PsMADS4. The length of PsMADS4 cDNA is 1081 bp including738 bp encoding frame, which encodes 245 amino acids. There are two conserved domains, MADS-box domain (3-75 amino acid) and K-box (89-176 amino acid). The study of PsMADS4-overexpression in Arabidopsis plants indicated that the blooming time of transgenic plants was earlier than that of wide plants, while the florescence of transgenic plants was a bit longer than that of wide plants. |
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