| ID1 is a classical flowering gene , which is expressed exclusively in immature leaves first reported in Zea mays. Late flowering and prolonged vegetative stages are typical phenotypes of id1 mutants. Its homologous genes have been found in several different plant species such as Oryza sativa, Sorghum bicolor. It was reported that this gene play an important role in the control of the transition from the vegetative to the reproductive phase in Zea mays and Oryza sativa, which is another pathway that is independed of photoperiod flowering pathway. Zea mays and Oryza sativa are classical short day plants, vernalization is not required for the transition of flowering. Triticum aestivum is long day plants, floral transition needs vernalization. It is not clear whether the similar molecular mechanism of floral transition exists in the Triticum aestivum. If the similar mechanism exist, it is interesting to study the relationship between the gene and the vernalization,in addition, whether the late flowering phenotype caused by loss-of-function ID1 mutants can be replaced by vernalization to promote flowering. We have tried to clone this gene in Triticum aestivum previously, but not successful, so we decide to use model plant Brachypodium distachyon to research. In this thesis, we select the Brachypodium distachyon Bd21 as experiment material, and a series of studies have been conducted on the physiological characteristics; isolation, sequence and expression analysis; tissue culture transformation system of Brachypodium distachyon, preliminary function identification of BdID1; in order to obtain some mutants revelant to flowering time and screen point mutation of BdID1 using Tilling technique, we used EMS mutagenesis seeds of Brachypodium distachyon Bd21, which can greatly help to study the function and mechanism of BdID1. The main results are as follows:1. Observation of growth of Brachypodium distachyon Bd21 in different vernalization and photoperiod conditions, we found that Brachypodium distachyon Bd21 is a weak winter, weak dormancy, long-day (LD) plants.2. We designed specific primers according to homologous sequences alignment, a novel gene, termed Brachypodium distachyon ID1 (BdID1), was isolated using RT-PCR and RACE-PCR from Brachypodium distachyon Bd21. The full-length cDNA of BdID1 is 1661bp, and encodes an 429 amino acid peptides, the length of 5'UTR and 3'UTR is 140bp and 231bp, respectively. Amino acid sequence alignment revealed that BdID1 shared high identity with the ID1 in Zea mays, Oryza sativa, Sorghum bicolor. The homology is from 56%-65%, it also contains a putative nuclear location signal, four zinc finge motifs and a conserved TRDFLG motifs. Two introns were found in the region of genomic sequence.3. Semi-quantitative RT-PCR revealed that BdID1 expression in the whole growth cycle, and simiarly to OsID1 show much broader expression. The BdID1 mRNA was more abundant in younger leaves than in the old leaves, root, stem , shoot apices.4. Explored Brachypodium distachyon tissue culture process, and tried to transformed pSQ5 binary expression vector, the combination of chemical selection (hygromycin) and visual screening (green fluorescence) drastically reduces the level of selectable agent compared with chemical selection alone. The screening of fluorescent sectors allows an early identification of transformation events. Construct sense, antisense and RNAi expression vector, and transformed them into the Brachypodium distachyon, hygromycin selection and the resistant transgenic seedlings obtained. 5. EMS mutagenesis Bd21 seeds, M2 generation of seeds have been harvested, several mutants phenotype related to flowering time have been obtained. |
