Cloning And Expression Of Floral Organ Development Related Genes In Tree Peony

Tree peony (Paeonia suffruticosa) is a very popular traditional famous flower and candiate national flower .It has high ornamental value. Molecular breeding has been one of the hotspots in flower type modification. Till now, although many genes and cDNA clones for genes about floral development have been isolated and well characterized, little information, if any, is available concerning molecular aspect of floral organ development in tree peony.In this study, we firstly isolated the full length cDNA clones encoding PsAP1, PsAP2, PsPI, PsMADS1, PsMADS5, PsAG and PsMADS5 from sepals, petals, stamens and carpels of tree peony cultivar'Zhao Fen'using RT-PCR and RACE, then, studied their expression patterns in different organs and stages using relative real-time PCR. Meanwhile, in order to validate the function of genes, the sense and antisense plant expression vectors of the seven genes were constructed and transformed into tobacco NC89, respectively. The main results were as follows:1. Using degenerated primers derived from conserved sequences of previously cloned genes, 7 genes involved in floral organ development were first obtained from sepals, petals, stamens and carpels of tree peony cultivar'Zhao Fen'by RT-PCR and RACE, and were named PsAP1 (GenBank No.HM14393), PsAP2 (GenBank No. HQ222889), PsPI(GenBank No.HQ878444),PsMADS1(GenBank No.HQ222890),PsMADS9 (GenBank No.HQ902183), PsAG(GenBank No.HQ222891) and PsMADS5(GenBank No.HQ449569), respectively. Sequence analysis showed that these genes all had MADS domains and the plant specific K domains,belonged to MADS-box gene family except PsAP2.2. The expressions of these seven genes were detected in different organs of tree peony by using relative real-time PCR. The results showed that different genes had different expression patterns and tissue specicity. PsAP1 mainly expressed in petals,sepals and carpels,but hardly expressed in roots and stems. PsAP2 expressed in all the organs,hinghest in petals. PsPI mainly expressed in petals and stamens, hardly expressed in any other organs. PsMADS1 and PsAMDS9 mainly expressed in petals stamens and carprls, hardly expressed in any other organs. PsAG mainly expressed in stamens and carpels, hardly expressed in roots and sepals. PsMADS5 expressed in all the organs, hinghest in petals.The expressions of these seven genes were detected in different stages of tree peony by using relative real-time PCR. The results indicated that different genes had different expression patterns. PsAP1 were expressioned at the highest level at prophase and the lowest level at anaphase in the process of flower bud differentiation. PsAP2 were expressioned at the highest level at prophase and the lowest level at the initial stage in the process of flower bud differentiation. PsPI PsMADS1 and PsMADS5 were all expressioned at the highest level at metaphase and the lowest level at the initial stage in the process of flower bud differentiation. PsMADS9 were expressioned at the highest level at anaphase and the lowest level at the initial stage in the process of flower bud differentiation. PsAG were expressioned at the highest level at terminal stage and the lowest level at the initial stage in the process of flower bud differentiation.3 The sense and antisense plant expression vectors of these seven genes(PsAP1,PsAP2, PsPI,PsMADS1,PsMADS9,PsAG,PsMADS5) were constructed and transformed into Arabidopsis thaliana and Nicotiana tabacum via Agrobacterium tumefaciens-mediated flower-dipping and leaf disk transformation method, respectively. Parts of transformed plants were obtained after genomic PCR detection.4. Over expression of PsAP1 in transgenic Arabidopsis thaliana and Nicotiana tabacum both resulted early flowering. It indicated preliminarily that PsAP1 plays an essential role in the regulation and control of flowering time.Over expression of PsAG in transgenic Arabidopsis thaliana, the transgenic plants appeared smaller leaves and abnormal flowers compared with wild type Arabidopsis thaliana. Over expression of PsAG in transgenic Nicotiana tabacum, the transgenic plants showed smaller flowers compared with wild type Nicotiana tabacum. It preliminarily indicated that PsAG may be related to flower development.