The Evidence Of β-actin And Nuclear Myosin Ⅰβ(NMⅠβ) In The Nuclei And Nuclear Matrices Of The Tobacco Suspension Cells

BY-2 tobacco leaves disinfected were cultured in the MS medium with 0.5mg/L 2,4-D and 0.5g/L casein. After 4-5 times subculture, the calli induced were light yellow, grainy and available for suspension culture. The calli were inoculated into the liquid medium of MS with 1mg/L 2,4-D and cultured on the rotatory shaker. After 4-5 times subculture, the suspension cells could be obtained. The cell wall of tobacco suspension cells was removed by cellulast and macerozyme. Dispersed protoplasts from suspension were achieved. So far, how to get the high purity plant nuclei was a challenge. According to the method from animals, organelles were isolated by lysing tobacco protoplasts in CSK buffer with 0.5% TritonX-100. Then, the purified nuclei were separated by differential velocity centrifugationand sucrose-percoll zonal centrifugation. The DNA of tobacco nuclei was stained with PI in cold and dark prior to flow cytometry analysis. The final concentration of PI was 50μg/mL. It proved that 83.41% of suspension cells cultured in normal were in G₀+G₁ phase. Then the nuclear matrices were obtained by digesting nuclei respectively with DNase I and high salt. The total nuclear proteins were extracted by nuclear-cytosol extraction kit. The results of the immunoblot of anti-β-actin antibody as immunomarker indicated that the cytoplasm, nuclei and nuclear matrices had positive reaction with the antibody. Similarly, immunoblot tests also showed that the nuclei reacted positively with the anti-NM I β antibody. The results of the immunoblot experiments verified the presence of β-actin was the constituent of the nuclei and nuclear matrices of the tobacco suspension cells. Simultaneously, NM I β was also a constituent of the nuclear proteins. As far as I known, it was the first time to report the evidence that the NM I β originated from the nuclei of the mouse fibroblasts located in the nuclei of the tobacco suspension cells. The author supposed the probable functions of β-actin and NM I β in the plant nuclei and nuclear matrices, meanwhile, inferred the conservation of the β-actin and NM I β during the biology evolution.