|其他摘要||As an important plant of the grass family, bamboo has special value in economy, ecology and society. Nowadays, according to some report, 50% of the total number of bamboo species in the world are close to die out, owing to bamboo flowering. Most of bamboo species flower only once in whole life after a long vegetative stage and die after that. The long vegetative growth as well as the low natural seed production made it uneasy to understand the mechanism of bamboo flowering. By developing in vitro tissue culture, bamboo flowering in vitro became possible. It allowed us to set up a bamboo in vitro flowering and flowering-reversion system, which paved a brand new way for species identification, breeding of improved bamboo, and production of interspecific-intergeneric hybrids, especially, providing a reliable source of bamboo seed which is a basis for studying the mechanism of bamboo flowering.
In this project, we focus on a tropical bamboo species, Dendrocalamus latiflorus in our study. On one hand, we tried to induce it to flower in vitro; on the other hand, we cloned an homolog gene of AtEMF2 from it, which is related to maintaining vegetative phase through repressing the flower program in Arabidopsis, and we explored its function by transgenic experiments, which offered important base for further study on bamboo flowering.
Study of in vitro flowering in Dendrocalamus latiflorus
In the past two decades, in vitro flowering of bamboo has been reported from many species in several laboratories of a few countries. It is allowed to set up a bamboo flowering and flowering-reversion system in vitro. In our experiment we used different kinds of cytokinins with different concentration to inducing shoots, propagating shoots and inducing flower buds step by step. Finally, we found an appropriate medium for the first two steps, and also induced flower buds in vitro at the third step. But restricted by the quantity of seeds, we did not find a good flowering line. It may need more seeds to carry on the study in the future.
Cloning and sequence analysis of DlEMF2 gene in Dendrocalamus latiflorus relating to bamboo flowering
Based on the conserved domain of EMF2 genes, we designed the degenerate primers and cloned the DlEMF2 gene from Dendrocalamus latiflorus. The complete cDNA of DlEMF2 was 2518 bp in length. Sequence analysis showed that it has three conserved regions, C2H2-type zinc finger motif, N-terminal basic domain and C-terminal acidic-W/M domain. In Arabidopsis, EMF2 protein complex belongs to Polycomb-group (Pc-G) proteins. In plants and animals the PcG proteins form multimeric epigenetic silencer complexes that control diverse developmental pathways. Polycomb group (PcG)–mediated gene maintains stably inherited repression of target genes to inhibit transcription which lead to gene scilencing.
Functional studies on DlEMF2 gene
Based on the cloning of DlEMF2 gene, we analyzed its expression among tissues including apical shoot, stem, leaf, root and inflorence by semi-quantitative RT-PCR. Its tissue-related expression pattern showed that the mRNA level of DlEMF2 was extensive in all parts of the plant and was higher in shoot, but it was much lower in inflorescence. Based on the protein sequence alignment, DlEMF2 has three conserved functional homolog regions as AtEMF2. In order to investigate the function of the DlEMF2 gene, we constructed anti-sense DlEMF2-C fusion construct, then transformed it into the Agrobacterium tumefaciens and subsequently into Arabidopsis. 59% of the transgenic plants showed earlier flowering with formation terminal flowers, others showed normal or unrelated phenotypes. Relative expression levels of endogenous AtEMF2 in these lines were tested by quantitative real-time PCR to confirm whether it has been regulated. The result confirmed that the exogenous DlEMF2 fragment was responsible for repressed endogenous EMF2 in Arabidopsis. Over expression of DlEMF2 in Arabidopsis showed late flowering in T1 generation which means there may be some differences in expression mode between DlEMF2 and AtEMF2. Rescue experiment showed that DlEMF2 can rescue the emf2-1, which means DlEMF2 has the same function as AtEMF2 in maintaining vegetative stage. A transient expression assay showed that the fusion protein of DlEMF2 and green fluorescent protein (GFP) was mainly located in the nuclei.|