Involvement Of Arabidopsis Thaliana Microtubule-binding Protein CLASP In The Regulation Of Mitosis Under Enhanced UV-B Radiation

Plants are stationary growing photosynthetic autotrophic organisms.Sunlight is the main energy source of plants.If plants need sunlight,they will inevitably be exposed to UV-B radiation,and increasing UV-B radiation will have many effects on plants,Such as morphological characteristics,physiological characteristics,genetic metabolism and other changes.CLASP(Cytoplasmic linker proteins,CLIP-associated proteins,CLASPs)are both microtubule-binding Proteins and Cytoplasmic junctions that regulate the morphology and orientation of microtubules during cell division.The loss of CLASP function can affect microtubule structure and function,cell division and enlargement,cell differentiation,cell morphogenesis,etc.In this study,the role of CLASP protein in cell mitosis was studied to explore the regulatory mechanism of the involvement of CLASP in mitosis in Arabidopsis under enhanced UV-B radiation.In this study,we used Arabidopsis thaliana and clasp-2 mutants as materials to compare the phenotypic differences between wild-type(WT)and clasp-2 functional loss mutants.The expression site and expression amount of CLASP were determined by p CLASP::GUS vector construction and the subcellular localization of CLASP was determined by the construction of p Super1300::CLASP-GFP overexpression vector.The control group and UV-B radiation group were set to statistically analyze the morphological characteristics,physiological and biochemical indexes,the expression level of CLASP gene and the expression level of cell cycle related genes of WT,clasp-2 mutant and overexpressed plants.The mitosis of Arabidopsis root tips was observed by modified phenolic fuchsin staining,and the cell division index and aberration rate were counted.The main research results are as follows:1.Clasp-2 homozygous mutants were identified.Compared with WT,clasp-2 mutants showed reduced germination rate,shorter root length,delayed mossing,shorter plant height and the number of trichome branches became less.2.pCLASP::GUS expression vector was successfully constructed and positive transgenic plants were obtained.The expression of CLASP in roots,stems and leaves of Arabidopsis thaliana was confirmed by GUS staining.3.Thep Super1300::CLASP-GFP overexpression vector was successfully constructed,and the positive transgenic plants were obtained by transient expression of tobacco and subcellular localization of overexpression lines in root tip,and the localization of CLASP in cytoplasm was confirmed.4.The morphological,physiological and biochemical indexes of WT,clasp-2 and transgenic plants after UV-B radiation were detected.The morphological changes of clasp-2after UV-B radiation were mainly as follows: decreased germination rate,shorter root length,shorter plant height,delayed mossing,and decreased proportion of superficial and The proportion of trichome branches decreased;Changes of physiological and biochemical indexes:MDA concentration increased,SOD activity decreased,chlorophyll content and soluble sugar content decreased.These results indicated that the growth and development of clasp-2 mutant were seriously affected by UV-B radiation.5.The detection of CLASP gene expression after UV-B radiation showed that the expression of CLASP decreased and was inhibited after UV-B radiation.6.Effects of UV-B radiation on the expression of cell cycle related genes:UV-B stress compared to WT control group,cell cycle related gene DKA1;1,CDKB2;1,CYCB1;1 and CDC5,the expression levels of were inhibited,and the inhibition was more obvious in clasp-2mutant.7.Effects of UV-B radiation on mitosis:Compared with the cell division index and aberration rate of WT control group,the cell division index and aberration rate increased when the cell division index was inhibited under UV-B radiation,while the cell division index and aberration rate of clasp-2 mutant were more significantly inhibited and the increase was more significant.In summary,this study analyzed plant morphological characteristics,physiological and biochemical characteristics,the expression level of CLASP gene,the expression level of cell cycle related genes,and the changes of mitotic cell division index and aberration rate.The role of microtubule-binding protein CLASP in the regulation of cell mitosis under UV-B stress in Arabidopsis thaliana was studied.The results showed that CLASP has a certain role in the regulation of cell mitosis under enhanced UV-B radiation.