| At present, GFP tag has been widely used in biology, and it has brought convenience to the observation in vitro, especially for observing intuitively the dynamics of the cytoskeleton.Microtubules play an irreplaceable role in the growth and development of plants. It not only plays an important role in the formation of plants, but also plays a very important role on cell division and microtubule nucleation through the interaction of tubulin and microtubule associated proteins in the plant.Microtubule associated protein GCP4 plays an essential role in the organization of microtubule nucleation, and promotes the interaction between ?-tubulin and microtubules in plants. In Arabidopsis, the absence of GCP4 gene led to the abnormal division phenomenon of abnormal spindle microtubule bundles and phragmoplast microtubule bundles reduction and dispersion. UV-B radiation, as one of the research hotspots of abiotic stress, has a widely influence on the biology. The low dose of UV-B radiation can regulate the growth of plants, but the high dose of UV-B radiation can damage the biology. Therefore, it will provide an importanttheoretical basis for the study of cell division and abnormal division for observing the effect of UV-B radiation on GCP4 and the dynamics of microtubule in vitro by using GFP tag fusion microtubule associated proteins GCP4.In this study, we used Arabidopsis thaliana as research object,and treatment Arabidopsis thaliana transgenic plants TUB-GFP and MBD-GFP through UV-B radiation, which the dose 4.53KJ?m-2?d-1is cumulated to 27.2 KJ?m-2, to observation and analysis. At the same time it will be fused to the GFP tag fusion GCP4 protein to construct GCP4-GFP expression vector by recombinant DNA technique of genetic engineering. The results showed that:1. Wild-type(Arabidopsis thaliana L., Col-0), TUB-GFP, and MBD-GFP plants have been treatment by UV-B radiation, which the dose is cumulated to 27.2 KJ?m-2. It found that it is slightly different between transgenic Arabidopsis and wild-type by UV-B radiation,and it has been showed significant shorter in plant height and root length in the TUB-GFP and MBD-GFP than wild-type. It indicated that it had effect on different physiological changes in different plants with UV-B treatment.2. TUB-GFP and MBD-GFP have been observed by using laser scanning confocal microscope with UV-B radiation treatment. And it found that microtubules in guard cells can be affected by different levels. UV-B treatment caused severe depolymerization of MTs in TUB-GFP. Guard cells were asymmetric, twisted and patched MTs were observed in stomata. At the same time, it was also obviously inhibited in the growth of TUB-GFP plants. Exposure to UV-B caused the appearance of spots and the depolymerization of MTs in guardcells of MBD-GFP, but it is more serious in TUB-GFP.3. Microtubule binding protein GCP4 acted as template, and connected to the cloning vector p UC19 by using double enzyme digestion method for sequencing. After successfully sequenced, the gene linked to expression vector p Super1300-GFP by double enzyme digestion method. After being verified through plasmid with PCR and enzyme digestion verification, we successfully constructed p Super1300-GCP4-GFP vector, and try to transfer recombinant vector into wild-type Arabidopsis thaliana by Agrobacterium mediated method.In summary, we used DNA recombinant technology to target gene and GFP tag fusion in this study and observed the dynamic of tubulin in vitro in response to UV-B radiation. It brings convenience to observe the effects on the plants by UV-B radiation. And it lays a theoretical foundation for the phenomenon of cell division and abnormal division. |
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