| sgf73 gene encodes Sgf73 protein subunit in SAGA complex,participates in heterochromatin assembly,and has transcriptional coactivator activity.psp3 gene encodes vacuolar serine protease Psp3,which is involved in the hydrolysis of vacuolar proteins.rpl1001 gene encodes ribosomal L10 e family proteins,which are involved in cytoplasmic translation,assembly of protein-containing complexes and assembly of large subunits in ribosomal biogenesis.According to the different functions of genes,the gene knockout may lead to the slow growth of the strains and the defects of chromosome,spindle and actin in mitosis.In this study,Schizosaccharomyces pombe was used to investigate the sporulation defects of fission yeast in meiosis under the conditions of nitrogen starvation in sgf73?,psp3? and rpl1001? strains.Fluorescence protein labeling and living cell imaging were used to study dynamic of chromosome,spindle and actin during mitosis.The experimental results were as follows:(1)The deletion of sgf73,psp3 and rpl1001 genes resulted in the slow growth of the strain,and the deletion of rpl1001 gene had a greater impact on the growth of the strain.The results of sgf73? and psp3? showed that there was significant difference between the the sgf73? and psp3? strain and wild-type after 2 h,and there was extremely significant difference after 6 h.rpl1001? showed significant difference with wild type after 6 h.(2)The number of spores of sgf73? and psp3? showed extremely significantly reduced after gene deletion,and psp3 gene had greater influence.The number of spores of rpl1001? showed increased extremely significantly,2.27 ± 0.23% produced 8 spores.(3)The microtubule statistics showed that the strains that produced 3 and 4microtubules during the interphase period after the sgf73 gene was deleted were extremely significant and significantly reduced,and the strains that produced 5microtubules increased significantly,56.67±10.41% produced 5 microtubules.(4)There was a long period of monopoly spindle and uneven chromosome segregation after sgf73 gene deletion.There was chromosome fragmentation,cell chain,spindle bending and spindle folding after the deletion of psp3 gene.There were chromosome fragmentation and uneven segregation appeared after the deletion of rpl1001 gene.The results showed that sgf73 gene knockout was not good for chromosome average,and psp3 and rpl1001 gene knockout was not good for chromosome integrity.(5)The results of the spindle elongation time showed that the sgf73 and psp3 gene knockout extremely significant prolonged the spindle elongation time.Among them,the psp3? strain took longer,and the rpl1001 gene knockout extremely significant shortened the spindle elongation time.The metaphase and anaphase of sgf73? was significant and extremely significant prolonged,respectively.The prophase and anaphase of psp3? were extremely significant and significant longer prolonged,respectively,and the metaphase and anaphase of rpl1001? were extremely significant shortened.The elongation rate of the spindle of the sgf73? decreased extremely significantly at anaphase,the elongation rate of the psp3? strain decreased significantly at prophase and anaphase,and the elongation rate of the rpl1001? strain increased extremely significantly at anaphase.In conclusion,the deletion of sgf73 and psp3 genes could lead to longer spindle elongation time and lower rate of spindle elongation.sgf73? was reflected at metaphase and anaphase,and psp3? strain was reflected at prophase and anaphase;while the rpl1001 gene deletion shortened the time of the strain at metaphase and anaphase and speeds up the rate of spindle elongation at anaphase.(6)The spindle statistics showed that the deletion of the sgf73 gene had a greater impact on the formation of the spindle,and the deletion of rpl1001? had a greater impact on the breakage of the spindle.Except for the bar spindle,45% and 30% of the sgf73?and psp3? strains formed dot and monopoly spindles,respectively,and 30% of the rpl1001? produced monopoly spindles.When the spindle was broken,41.67%±2.89 and81.67±2.89% of the psp3? and rpl1001? strains were linear-type breakage,respectively.The sgf73? strain had no significant change in spindle breakage.(7)The analysis of cell morphology showed that the deletion of psp3 gene has a greater impact on cell morphology.The cell length of the sgf73? was significantly different from wild-type at each stage of mitosis.The cell breadth of the sgf73? was significantly and extremely different from the wild type at multiple time points,and the cell length-breadth ratio at the end of division is extremely significant from the wild type.The cell length and width of the psp3? strain was significantly different from the wild type at each point of mitosis.The rpl1001? strain was significantly different from the wild-type cell length at the end of division,and was extremely significant different from the wild-type cell length at other points.Moreover,the cell breadth of rpl1001? strain at each time point was significantly different from that of the wild type,and the cell lengthbreadth ratio at the end of division was extremely different from that of the wild type.(7)The retention time of the actin contraction loop in sgf73?,psp3?,and rpl1001?was extremely significant different from the wild-type.The contraction time of the actin contraction loop of the sgf73? and psp3? was significantly different from the wild-type.And very significant difference in which psp3? actin stays and contracts longer than sgf73? and rpl1001?.At the same time,the contraction loop rate of actin in the sgf73?and psp3? was extremely significantly different from the wild-type.This shows that psp3 gene knockout has a greater impact on the contraction loop of actin.This study revealed the changes and defects of cell morphology and dynamics of chromosome,spindle and actin after sgf73,psp3 and rpl1001 gene knockout respectively.At the same time,this study provides a theoretical basis for specific mechanism of cell dynamics of chromosome,spindle and actin and human to conquer related diseases. |
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