Effect Of Enhanced UV-B Radiation On Microtubule Associated Protein MAP65-1 In Arabidopsis

With the rapid development of science and technology in the world,the aspects of environmental crisis are becoming more and more extensive,especially the serious destruction of atmospheric environment,making the ozone layer thinning,resulting in the enhancement of UV-B radiation reaching the surface of the earth.It seriously affects plant morphogenesis,genetic characteristics,growth cycle and other life activities.MAP65-1 is a unique microtubule associated protein,which plays a key role in the dynamic assembly of microtubules.It can stabilize microtubules and make microtubules bundle.To study its involvement in abnormal mitosis in response to UV-B radiation,The aim of this study was to explain the molecular mechanism of the effects of enhanced UV-B irradiation on cell mitosis and to provide a theoretical basis for improving the response of plants to UV-B stress.In this study,the roots and leaves of Arabidopsis thaliana seedlings were treated with UV-B radiation.After morphological observation,the overexpression vector of At MAP65-1 gene was constructed,and the homozygosity of T-DNA inserted mutant was identified.The effect of map65-1 in response to UV-B radiation was studied,and the main results were as follows:(1)Bioinformatics was used to predict the open reading frame,hydrophilicity,,signal peptide and subcellular localization of MAP65-1 protein.The results showed that the open reading frame of At MAP65-1 was 1764 bp,encoding a total of 587 amino acids,and the subcells were located in the nucleus or cytoplasm.The isoelectric point is 4.97 and the molecular weight is 65.78 k Da,which contains 62 phosphorylation sites and belongs to hydrophilic non-secretory protein.(2)At MAP65-1 gene overexpression vector p GFPXB-4-MAP65-1 was constructed and introduced into tobacco leaves by Agrobacterium tumefaciens.The transient expression of the fusion protein in tobacco leaves was observed under confocal laser microscope,which proved that the vector was successfully constructed.(3)Overexpression vector was used to transform wild-type Arabidopsis thaliana by Agrobacterium tumefaciens-mediated flower soaking method.The overexpression lines of At MAP65-1 gene were successfully screened from DNA level,transcription level and cell level of Arabidopsis thaliana.(4)At MAP65-1 gene T-DNA inserted into homozygous mutants was obtained by double primer method and semi-quantitative RT-PCR method.(5)In the condition of enhanced UV-B radiation,the determination of the root length,plant height,MDA,soluble sugar content soluble protein and antioxidant enzyme activity of Arabidopsis map65-1 mutants and MAP65-1 over-expression line showed that mutants were sensitive to UV-B radiation,followed by over-expression lines,The result shows that map65-1 protein may play a role in response to UV-B radiation.(6)Semi-quantitative RT-PCR and Laser confocal microscopy(MAP65-1)in different treatment groups showed that UV-B radiation inhibited the expression of map65-1.