Effects Of Enhanced UV-B Radiation On EB1c Of Arabidopsis

Since the 20 th century,with the emissions of chlorofluorocarbons and other nitrides into the air have been increased,resulting in the ozone layer thinning and the emergence of an ozone hole,UV-B(ultraviolet-B)radiation in the sunlight reaching the ground also increased.UV-B radiation increased influence in many ways to the growth and physiology of plants.In this process,UV-B radiation is not only an environmental stress factor,but also an important signal regulator.EB1(End Binding Protein 1)is about 32 KD,it is one of the microtubule-assiciated proteins(MAPs),which is evolutionarily conserved and located at the positive end of the growing microtubule.EB1 plays an important role in regulating the dynamics of microtubules and recruiting other interactional tubules.EB1 family members of Arabidopsis have EB1 a,EB1b and EB1c;EB1a and EB1 b form heterodimers with each other,however,EB1 c is not involved.There is a nuclear locialization signal(NLS)at the C-terminal of EB1 c,which plays an important role in the regulation of mitotic spindle localization and chromosome segregation.In order to study the response mechanism of EB1 c under the condition of UV-B radiation enhanced,three kinds of Arabidopsis are used as materials:wild type(WT)plants,mutant(eb1c)plants and transgenic(EB1c:GFP)plants.The purchased eb1 c mutant was identified by "three primer method" at the DNA level,the homozygous eb1 c mutant was obtained by further validation at RNA level with actin as internal reference.Compared with wild type,it was found that the root length of the mutant was shorter and the difference was not significant,bolting was earlier than wild type,and there was no significant difference between the two plants of the pod length and plant heigh.The recombinant plasmid of psupper1300-EB1c-GFP was successfully constructed in the early study of our laboratory was used as the material and was re-sequenced to verify theconstruction was successful,then it was expressed transiently in protoplasts of Arabidopsis and epidermal cells of tobacco.The green fluorescence of GFP was observed in both transient expression systems,it shows that the EB1c:GFP fusion protein was successfully expressed,it was further proved that the recombinant plasmid was successfully constructed.In the transient expression of tobacco epidermal cells,chromosomes were stained with DAPI,the EB1c:GFP fusion protein was the same as the chromosome localization,indicating that EB1 c was located in nucleus of tobacco epidermal cells.The homozygous transgenic plants of T3 generation were obtained by dipping flowers into Arabidopsis by the recombinant plasmids,and screened with Hyg.Compared with wild-type,there was no significant difference at the root length of one week old plants,but the difference was significant of two week old plants.The bolting was not significant.Pod length and plant height of transgenic plants are significantly higher than wild-type plants.The expression of EB1c:GFP fusion protein in tissues of Arabidopsis was observed,it shows that EB1 c was successfully expressed in root tips and stomatas,recombinant plasmid was successfully constructed and expressed in Arabidopsis was further proved.In order to study the effects of enhanced UV-B radiation on EB1 c,the root length,the content of soluble sugar,soluble protein and MDA were compared under normal growth condition and UV-B radiation condition.Mitotic cells in the apical meristem of Arabidopsis were also observed during anaphase and telophase.The results shows that the root length,soluble sugar content,soluble protein content and MDA content of eb1 c group were all the most sensitive to enhanced UV-B radiation,the WT group was the weaker,the EB1c:GFP group was the weakest.On the condition of strong UV-B radiation,the cells of the root apex of the eb1 c group showed abnormal mitosis in thetelophase,while the abnormal division of the chromosomes was not found in WT and EB1c:GFP group.However,in the mitotic interphase and anaphase EB1c:GFP fusion protein was colocated with the chromosomes.The results indicated that abnormal division of chromosome in mitotic anaphase of the mutant plant was probably related to the deletion of EB1 c gene function under the condition of enhanced UV-B radiation.