Expression And Functional Analyses Of Vegetative To Reproductive Transition2Gene In Brachypodium Distachyon(L.)

The transition from vegetative to reproductive growth is one of major developmental transitions in the life cycle of plants. The wheat (Triticun aestivum L.) is one of the most important grain crops in the world. The flowering time is tightly controlled by complex genetic regulatory networks in response to endogenous and environmental flowering signals. At present, the analysis of wheat flowering time has some diffculties, because the genome is large, transformation is diffcult and so on. In2001, Brachypodium was proposed as a model system for the study of the grass family, the reason is that its genome is small, rapid generation time, self-fertile, a high efficiency transformation, particularly, a close relationship with wheat. In this research, two novel BdVRT2genes were isolated from Brachypodium distachyon. Through Agrobacterium-mediated transformation of Brachypodium distachyon Bd21to obtain transgenic plants, we examined the expression pattern and biological function of BdVRT2and BdVRT2-like, the results are as follows:(1) The analysis of the structure of the genome showed that there are eight exons and seven introns in the genome of BdVRT2. The length of the BdVRT2cDNA is675bp, encoding225amino acid. BdVRT2is more closely related to TaVRT2(85.84%amino acid sequence identity) than BdVRT2-like (77.39%amino acid sequence identity); BdVRT2-like contains eight exons and eight introns in the genome with an open reading frame of690bp encoding a putative protein of230amino acids. It is more closely related to ZmM19(89.96%amino acid sequence identity).(2) The molecular characteristic and phylogenetic analysis revealed that BdVRT2and BdVRT2-like genes are belonging to MADS-box gene family which is associated with flowering time. And the two genes homology is up to77.39%, the region in the MADS-box and C-terminal is highly homologous while the K-box is relatively specific. That may suggest the differences function in the target genes. (3)Day length influences the expression of BdVRT2, The expression patterns of BdVRT2indicated that its transcripts are abundant Before the spikelet formation during long-day conditions, indicating that BdVRT2plays a role as a repressor of the floral transition in Brachypodium, However, under short-day conditions the expression levels of BdVRT2gradually improved along with the growth of plants; All that proved that the expression of BdVRT2was regulated by photoperiod. And BdVRT2-like is influenced by long-day, but is not affected by short-day.(4) The expression analysis of BdVRT2in the vernalization conditions show that BdVRT2were induced by cold, during the vernalization, BdVRT2mRNA is rather constitutively expressed, although there is a distinctively increased by7d of cold, suggesting its possible role in the vernalization. We also observed slight but significant upregulation of BdVRT2-like mRNA during vernalizing cold, but its function is still unknown so far. Therefore, BdVRT2and BdVRT2-like are considered to be induced as a result of vernalization.(5) BdVRT2expression exhibit a diurnal change, the expression analysis of BdVRT2about circadian rhythm shows that under long-day or short-day conditions BdVRT2expression levels were displayed a strong diurnal rhythm, Maximal expression levels were observed at the end of the light period and expression decreased with the onset of darkness, indicating that expression of BdVRT2has some circadian rhythm. And BdVRT2-like is not considered to be regulated by circadian rhythm because no significant change in their mRNA expressions.(6) The expression pattern of BdVRT2was studied by Real time RT-RCR. The abundance of BdVRT2was examined in various tissues and it was found to be predominantly expressed in shoot apical meristem and low in late reproductive tissues and roots. BdVRT2-like expression in the old leaves and shoot apical meristem was higher than in other tissues or organs during long-day, Moreover, BdVRT2-like transcripts were detectable mainly in the sheath and shoot apical meristem under short-day conditions.(7) To investigate the potential role for BdVRT2gene in flowering time function, The TO BdVRT2over-expressing plants showed a pleiotropic phenotype with altered shoot architecture and delayed flowering, the phenotype of transgenic plants over-expressing BdVRT2demonstrated that BdVRT2may regulate the floral transition, which may be a repressor of the floral transition in Brachypodium, on the other hand, morphology and phyllotaxy on the inflorescence meristem appeared abnormal, the rachis internodes were elongated giving the spike a different phenotype compared with wild type, in the weak type, transgenic plants does not affect the development of the floral organ which can gain seeds. However, in the strongest lines, the developmental delay was such that the spike did not form any floral organs and dried out.