【作者】 李冰霞；

【导师】 罗琛；

【作者基本信息】 湖南师范大学 ， 动物学， 2002， 硕士

【摘要】 本文用组织切片方法系统地观察了用被紫外线照射的湘江野鲤(Cyprinus carpio xiang jiang)的精子作激活源启动草鱼(Ctenopharyngodon idellus)卵子雌核发育的细胞学变化过程。确定了在一定孵化温度下，被激活的草鱼卵子完成第一次染色体复制的准确时间，实施人工染色体加倍处理的最佳时机，人工加倍处理的最佳方法和处理强度等。为通过抑制第一次卵裂，得到高比例基因纯合型雌核发育草鱼提供了实验依据。并得到了在遗传育种理论研究和生产应用上均有重要价值的基因纯合型的雌核发育草鱼。 实验结果表明：在24℃孵化水温下，草鱼卵在被激活后21min处于第一次卵裂的S期；24min进入第一次卵裂前期；27-30min处于中期；33min进入后期；36min处于末期。由此可知被激活的草鱼卵子在第27min时已经完成染色体的复制，使草鱼卵子雌核染色体人工加倍的最佳时期是在被激活后的27-30min这一时间区段内。 用不同的温度处理完成了染色体复制的被激活卵，以确定抑制被激活卵的第一次卵裂的最适休克温度。切片分析的结果表明在39℃以下的水温处理2min的被激活卵，有丝分裂器完全不被破坏，说明39℃以下水温对草鱼卵的热休克处理是无效的。在40℃水温处理2min的被激活卵，有丝分裂器部分被破坏，但这种破坏是不完全的。在41℃、42℃水温处理2min的被激活卵，有丝分裂器完全被破坏。但在42℃处理的胚胎死亡率很高。所以在41℃处理是使染色体组加倍的最佳热休克温度。而在41℃处理2min是比较合适的热休克强度。 观察解除热休克后被处理卵子纺锤体恢复情况，发现有72.8％的激活卵在解除休克后6min内恢复有丝分裂器，27.2％的卵子内未出现纺锤丝和纺锤体。未恢复纺锤体的激活卵在解除休克后6min，卵内染色体开始核化，9min时形成新的二倍体细胞核，14min时处入再次分裂的前期，19min时进入分裂中期，24min进入分裂后期，29min时处于分裂末期，且可以看到子核的形成。 此外，对雌核发育草鱼细胞遗传学分析证实所得雌核发育草鱼的染色体数目为48条，其核型公式为18m+2 4sln+6St;臂数为哪习O。未发现外源鲤鱼精子可能带入的额外染色体或染色体片断。说明这些草鱼是经过染色体加倍所得的、完全真实的雌核发育二倍体草鱼。更多还原

【Abstract】 The developmental procedure of first cleavage of grass carp eggs activated by UV-irradiated common carp sperm were systematically observed by method of histological section. The optimum parameters, such as the exact time for the activated eggs finishing the first chromosome sets copying, the optimum period for artificial chromosome set duplication, the optimum temperature and intensity of heat shocking etc, were determined. These parameters provide scientific basis for developing the technique of producing high rate of homozygous diploid mitogynogenetic grass carp. Indeed, groups of homozygous diploid mitogynogenetic fry of grass carp, which is very important for both the theoretical and practical research of genetic breeding, have been produced with this techniques based on the parameters.Results show that when incubated at 24, the activated eggs entered the S phase after activation for 21 min, the prophase for 24 min, the metaphase for 27-30 min, the anaphase for 33 min and the telophase for 36 min. Obviously, the optimum period for chromosome set duplication is from 27 to 30 min after the eggs were activated, since the chromosome set of the activated grass carp eggs was copied completely at that time.In order to define the optimum temperature for inhibiting the eggs’ first cleavage, different temperatures were tested to heat shock the activated eggs that had finished the chromosomeset copying completely. Results showed that the treatment is invalid by the heat shocking below 39 for 2 min, because no spindles of the activated eggs were destroyed by these treatments. When the activated eggs were treated by the heat shock at 40 for 2min, the spindle was partly destroyed. Only by over 41 ,the spindles were destroyed completely. But the mortality rate of embryos with the treatment of 42 was too high. So the optimum temperature for chromosome set duplication is 41 癈 and the effective intensity of treatment of heat shocking is 41 for 2 min.After the heat shocking was released, the spindles reformed in about 72. 8% of the observed activated eggs and did not in the others. After the treatment was removed for 6 min, the nucleus began to reform in those eggs that the spindle did not recover. After 9 min, new nuclei had been reformed completely. The activated eggs entered the prophase of the next cleavage cycle in about 14 min, the metaphase in 19min; the anaphase in 24min and the telophase in 29min.Cytological genetic analysis revealed that the chromosome number of gynogenetic grass carp is 48 and the karyotype is 18m+24sm+6st, fundamental arm number (NF) is 90, just as those of the common grass carp. No additional chromosomes or chromosomal fragment were observed. This result mean that the fish obtained by the above technique treatments is real diploid mito-gynogenetic grass carp.更多还原