| Since the 20 th century with the continuous advancement of human industrialization,the increase of nitrogen oxides such as chlorofluorocarbons discharged into the air has led to the continuous thinning of the ozone layer on the earth's surface in the sunlight reaching the earth's sueface.Ultraviolet B is also enhanced.Enhancing UV-B can have serious impact on the physiological growth of plants and animals on the Earth's surface.The chromatin assembly factor FAS1 is involved in the function of histones H3 and H4,and affect DNA synthesis and participating in cell mitosis.In this study,the chromatin assembly factor FAS1 was used as the entry point to study the molecular mechanism of its involvement in the abnormal mitosis generated by UV-B radiation,which would provid a theoretical basis for studying plant responsing to UV-B radiation.The wild type and fas1 mutant Arabidopsis thaliana were used as materials to construct pSuper1300:FAS1-GFP overexpression vector and pFAS1::FAS1-GFP self-promoter expression vector,respectively,overexpressing lines,restoring expression lines and T-DNA insertion mutant homozygous strains were identified to observe the morphological characteristics of different plants after UV-B radiation,and the changes of physiological and biochemical parameters were determined.The location of FAS1 and chromosomes was observed by confocal laser scanning micorscope microscopy.Thefound that FAS1 responsed to UV-B radiation was analyzed.The possible mechanisms of abnormal mitosis are produced.The main results are as follows:(1)Constructing pSuper1300:FAS1-GFP overexpression vector,pFAS1::FAS1-GFP self-promoter vector,introducing pSuper1300:FAS1-GFP overexpression vector recombinant plasmid into tobacco epidermal cells by Agrobacterium GV3101 infection method,and observing GFP-containing label under confocal laser scanning micorscope microscopy.The transient expression of the fusion protein in tobacco leaves demonstrated the successful construction of the vector and determined that FAS1 co-localizes with the chromosome in the nucleus and cytoplasm.(2)A homozygous mutant of the T-DNA insertion of the FAS1 gene was identified using the "three primers" method.(3)Overexpression vector and self-promoter vector were transformed into wild-type Arabidopsis thaliana plants and homozygous mutant Arabidopsis thaliana plants by Agrobacterium immersion method,respectively,and homozygous transgenic plants were screened with hygromycin,and DNA was obtained from DNA and RNA levels were identified in positive transgenic plants,and Arabidopsis thaliana plants were successfully screened for expression and Arabidopsis thaliana plants were restored.(4)Compared with wild-type Arabidopsis thaliana and mutant Arabidopsis thaliana,the root length of the 7-day-old mutant was found to be shorter than that of the wild type,and the difference was significant.The mutant Arabidopsis thaliana migrated from the vegetative growth stage to the reproductive growth stage earlier than the wild type Arabidopsis thaliana.There were significant differences in the ripe fruit pods and plant heights of the two plants.(5)Compared with wild-type Arabidopsis thaliana and over-expressing Arabidopsis thaliana,it was found that there was no significant difference in root length of 7-day-old plants.The length of the mature fruit pods and the plant height transgenic plants were significantly higher than the wild type plants,and the difference was significant.(6)In order to study the effects of UV-B radiation on plants,the experimental design was divided into four treatment groups,WT plants,pSuper1300:FAS1-GFPoverexpressing plants,fas1 mutant plants,and pFAS1::FAS1-GFP to restore expression plants.The root length,plant height,soluble sugar,soluble protein and MDA content were detected under normal light and UV-B radiation conditions.The results showed that the root length,plant height,soluble sugar,soluble protein and MDA content of fas1 mutant Arabidopsis thaliana were most sensitive to UV-B radiation,and WT plants and pFAS1::FAS1-GFP restored the expression of plants to a similar extent,pSuper1300:FAS1-GFP overexpressing plants were least sensitive to UV-B radiation.(7)In order to study the correlation between FAS1 and chromosomes during UV-B radiation treatment,the Arabidopsis root tip was labeled with DAPI in the experiment,and the chromosome and cell division phases were observed by confocal laser scanning micorscope microscopy.FAS1 was concentrated in the nucleus;In the case of UV-B radiation,no significant abnormal splitting was observed.Chromatin assembly factor is a highly conserved heterotrimeric complex that plays an important role in the growth and development of plants and animals.Chromatin assembly factors ensure the stability of heterochromatin in cells and keep related genes silent.By studying the enhancement of UV-B radiation,wild-type plants,fas1 mutant plants and the dynamic changes of chromosomes and FAS1 during mitosis in root tip cells of transgenic plants,it provides a further theoretical basis for revealing the molecular mechanism of "Splitting split" phenomenon. |
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