Dynamic Change Analyses Of Centromeres And Telomeres In Fusion-fission Cycles Of Mulberry Chromosomes (Morus Notabilis)

Mulberry is a perennial woody plant belonging to Rosales,Moraceae,Morus L.,Morus spp..Mulberry has important economic and medicinal values and has been cultivated in China for at least 5,000 years.As the main origins of mulberry,China has the largest distribution of mulberry species and the highest abundance of resources.The genetic background of mulberry is complex and diverse because of the long and complicated process of artificial breeding.Due to the fact that the chromosomes of mulberry are most dot chromosomes(except Morus notabilis and M.yunnanensis);it is difficult to prepare mulberry chromosomes.In the previous study,researchers first reported the chromosome fusion-fission cycle phenomenon in the process of generation alternation within individuals of mulberry.The fusion and fission occurred at the ends of chromosome 5 and chromosome 7,which could be located by 25 S rDNA probe.In order to explore the specific mechanism,the Arabidopsis-type telomere probe At-tel and 25 S rDNA of mulberry probe were used.The chromatin fiber fluorescence in situ hybridization(fiber-FISH)analysis was performed to analyze the fusion-fission sites of M.notabilis chromosome to obtain a higher resolution of the sequence.Through the co-localization of centromere probe Mn-175 and centromere-specific histone Mn CENH3 antibody,telomere probe At-tel and telomere binding proteins(Mn TRB1,Mn POT1 a and Mn POT1b)antibodies in different cell division stages of mulberry chromosomes,the dynamic changes of centromeres and telomeres in the process of fusion-fission cycle were tracked and analyzed,and the mechanism of chromosome fusion-fission cycle in mulberry was tried to be interpreted.It provided data support for molecular cytogenetics of mulberry.The main results were as follows:1.Fiber-FISH of fusion-fission site of chromosome in M.notabilis Using 25 S rDNA probe and telomere probe At-tel,the finer distribution of chromosome sequences at the fusion-fission site was obtained.In the co-localization region of 25 S rDNA probe and telomere probe At-tel,most chromatin fibers were covered by 25 S rDNA probe,and signals of telomere probe At-tel were mainly distributed as short and scattered dot signals,likely to reflect interstitial telomere sequences.Therefore,it was speculated that:(1)there were a few Arabidopsis-type telomere repeats(typical plant telomere repeat)near the fusion-fission site,but not large number of Arabidopsis-type telomere repeats;(2)there may be other special telomere sequences around the fusion-fission site,which can be localized by the 25 S rDNA probes on chromosomes 5 and 7.2.Immunofluorescence analysis of centromeres and telomere binding proteins in M.notabilisThree telomere binding proteins,Mn TRB1,Mn POT1 a and Mn POT1 b were screened from the genomic database of M.notabilis via bioinformatics,and three purified proteins were used to prepare antibodies.Antibodies to Mn CENH3 were already prepared in our laboratory.The position changes of centromeres and telomeres in different periods of meiosis and in metaphase of mitosis were traced by immunofluorescence assay with four protein antibodies.In terms of centromeres,Mn CENH3 antibody signals covered most chromatin regions throughout meiosis,and there were 1-2 strong signal spots in each cell item.However,in the metaphase of mitosis,the difference of Mn CENH3 fluorescence signal was more obvious: among the 12 chromosomes,there was one pair chromosome with no fluorescence signal,and there was another pair chromosome with only weak green fluorescence signal,strong fluorescence signal was clearly observed on 3chromosomes.Even in the same period,the activity of centromeres on different chromosomes was imparity.It suggested that the mechanism of chromosome fusion-fission cycle may be related to centromeres.Different from Mn CENH3,about telomere binding proteins,the immunofluorescence results of Mn TRB1,Mn POT1 a and Mn POT1 b proteins were not good enough.The immunofluorescence results in different periods of meiosis were not ideal,and only weak signals could be observed,and the number and location of fluorescence signals were not stable.It resulted to the failure to use antibodies against Mn TRB1,Mn POT1 a and Mn POT1 b proteins to track the changes in different periods.This indicates that the prepared telomere-binding protein antibody cannot be used to track telomere location.On the one hand,the valences of antibodies of 3 prepared telomere binding proteins were too low to track telomere location.On the other hand,the expression abundance of the above three proteins may also be one of the reasons affecting the immunofluorescence signal.Further experiments,such as protein expression analysis or optimization of experimental system,were still needed to analyze and troubleshoot the causes of unsatisfactory experimental results.3.The centromeres and telomere binding proteins constructed fusion proteins with EGFP for localizationThe EGFP fusion protein expression vectors of Mn CENH3,Mn TRB1,Mn POT1 a and Mn POT1 b were constructed and infected by Agrobacterium into mulberry plants.After successful expression,the positions of the EGFP fusion protein expression vectors were tracked,so as to analysis the position changes of centromeres and telomeres in different periods of chromosome fusion-fission cycle.Four EGFP fusion proteins were successfully expressed in mulberry root tips.Compared with the control group,the root tips of experimental group showed obvious green fluorescence.Chromosomes were prepared with root tips that successfully expressed EGFP fusion protein.After FISH experiment using probe Mn-175 and At-tel,it was found that the fluorescence of EGFP was very weak,and it was difficult to obtain convincing experimental results for indicating the exact position of telomeres and analyzing the changes of telomeres in different periods.More effective results could not be obtained after repeated experiments.Maybe the instability of experimental system caused degeneration of EGFP fusion protein and loss of fluorescence label.In this study,the location of centromere of Mulberry was tracked in multiple cell stages by combining nucleic acid probe Mn-175 and specific protein Mn CENH3 antibody for the first time.Three telomere binding proteins,Mn TRB1,Mn POT1 a and Mn POT1 b,were screened and identified,antibodies were prepared,and the positions of telomere in different periods were tracked.Although not satisfactory results were obtained,the results provided experimental basis for subsequent studies on telomere of mulberry.In this study,EGFP fusion protein was also successfully expressed in mulberry root tip.Although the subsequent FISH experiment did not achieve ideal results,it provided experimental data basis and prospects for future research on centromeres and telomere related proteins of mulberry.Fluorescence in situ hybridization experiments were difficult,especially for the woody plant M.notabilis,there were time and season constraints to obtain materials of different periods.The combination of fluorescence in situ hybridization and immunohistochemistry techniques to demonstrate the expression signals of centromeres and telomere related proteins further increases the difficulty of the experiment.Although this study failed to obtain high resolution research results,it is still a feasible attempt.