Function Analysis Of Cryptochromel In Subcell And Construction Of An Activation Tagging Library Of Arabidopsis

Plant growth and development process are affected by many external factors.Light as a major environmental element, not only provides the energy required for photosynthesis, but also plays an important role in photomorphogenesis. The evolution of higher plants has a fine feeling of light and signal system, to monitor the direction of light signal, quantity and quality to adjust their growth and development. A large number of studies have shown that blue light receptor cryptochrome-1 mediated blue light to regulate plant growth and development, including the inhibition of hypocotyl elongation, cotyledon opening, stomatal opening, anthocyanin accumulation and the regulation of gene expression. With further research, the study of CRY1 biological functions in subcellular, isolation and identification of Up-and down-stream components was very essential to understand the molecular mechanism of CRY mediated light signal pathway. In this work, we used different lights to analyse subcellular localization of CRY1 and light-induced migration process. On this basis, we constructed the CRY1-GR/cry-304 transgenetic plant on cry1-304 background. Control the location of CRY1 in subcell with the glucocorticoid to assess the biological functions. At the same time, we also contructed the activation tag mutant library on cry1cry2 background, isolated and identified the factors associated cryptochrome components. Through biochemical, genetic and other analysis methods, main results were obtained as followed:1. The 35S:: CRY1-GFP transgenic plants were grown for 7 days in the dark condition, and then dealt with different lights. The results showed that no GFP fluorescence in the dark. Under blue light, GFP fluorescence gradually increased with intensity increasing of the blue light, and expressed in cytoplasm and cell membrane, and had a transfer from cytoplasm to nucleus. As the treatment of blue light during 0-15 min, GFP fluorescence intensity was the highest, and the nuclei and cell GFP fluorescence intensity ratio reached a peak, then the GFP fluorescence was gradually quenched. Western blot results showed that the expression of GFP protein being directly proportional to the intensity of blue light, but not red light.2. The wild type were grown for 7 days in the dark condition, then dealt with different light qualities for 30 minutes and nuclear protein was extracted. Western blot analysis showed that: the abundance of blue-induced CRY1 protein was higher than it in red light and dark. The results proved the CRY1 protein specifically induced by blue light, and transported from the cytoplasm to nucleus.3. We obtained CRY1-GR/cry1-304 transgenic plants on the cry1-304 mutant background, and screened multiple independent lines. The results showed that, CRY1-GR/cry1-304 transgenic plants restore WT phenotype only in the absence of blue light and glucocorticoid (+Dex) conditions, including inhibition of hypocotyl elongation, cotyledon opening, anthocyanin accumulation and stomatal opening. The results show that: Nuclear localization signal (NLS) and photon activation are sufficient and necessary for CRY1 to play function in nucleus. The present study found no evidence of a role for cytoplasmic CRY1 in the control of hypocotyl, stomatal opening and cotyledon petiole elongation, but cotyledon expansion, anthocyanin accumulation all displayed a dependence on cytoplasmic CRY1.4. We got a gain-of-function mutant library on cry1cry2 background and screened nearly 2000 independent transformant lines. 35 transformant lines had obvious phenotype, and some of the mutant showed delayed flowering time. We also defined the flanking sequence of T-DNA insertion site in Scc101-D mutant through I-PCR analysis. The results showed that the phenotype of Scc101-D mutant caused by the overexpression of At5g28237 gene.5. The At5g28237 overexpression transgenic plant can restore Scc101-D mutant phenotype of delayed flowering, this result confirmed that the phenotype of Scc101-D mutant caused by the At5g28237 gene overexpressed. At5g28237 gene was tryptophan synthase gene belongs to the family ofβ-subunit. And the gene gun bombardment of onion skin experiments show this gene was localized in the cytoplasm, and no tissue-specific expression. At5g28237 transgenic plant growed slowly, and exogenous tryptophan can restore wild type phenotype. The phenotype of At5g28237 transgenic plant was similar with At5g58470 deficient mutant. According to the structure of amino acid sequence analysis, the salt bridge formation between the two sites and substrate binding sites are different. Therefore, we speculated that in Arabidopsis thaliana At5g28237 gene may not exercise TRP synthase beta subunit function. But over-expression, it competed At5g58470 protein complex in the tryptophan synthase locus body, the resulting loss of the At5g58470 similar phenotype. In addition, the transcription levels of FT and CO were significantly reduced in At5g28237 transgenic plant, comfirming their involvement in the regulation of flowering signaling pathways.