【作者】 孙远东；

【导师】 刘少军；

【作者基本信息】 湖南师范大学 ， 动物学， 2003， 硕士

【摘要】 异源四倍体鲫鲤是世界上首例人工培育的两性可育的四倍体鱼，这为研究四倍体的生物学特性提供了良好的实验材料，本文对异源四倍体鲫鲤的遗传特性进行了研究，主要包括其遗传稳定性、雌核发育、雄核发育方面的研究。 1．异源四倍体鲫鲤F₉-F₁₂染色体和性腺观察 采用肾细胞染色体制片技术，检测了异源四倍体鲤鲫F₉—F₁₂代的染色体数目，证明F₉—F₁₂继续保持四倍体性，染色体数目为4n=200条；观察了F₉—F₁₂的成熟性腺。在这三代四倍体鱼中它们仍然保持有正常的卵巢和精巢，能够分别形成正常的二倍体卵子和二倍体精子。用超薄切片技术，观察了四倍体鱼成熟卵巢和精巢的结构，并对成熟的卵子和精子的结构进行了观察，成熟的精巢由许多小叶组成，小叶内可观察到精源细胞、精子细胞、和成熟的精子，二倍体精子由头部和尾部组成，头部充满致密染色质，尾部中央轴丝具有典型的“9+2”微管结构；成熟卵巢中的Ⅳ时相和Ⅴ时相卵母细胞的细胞质内伸出许多微绒毛进入透明带内，并穿过厚厚的卵黄膜一直深入到细胞质中，推断微绒毛具有物质和信息交流的功能，Ⅳ时相和Ⅴ时相卵母细内的卵黄具有两种形式，一种其外有囊泡包围，另一种没有。在自然环境下，观察了异源四倍体鲫鲤自行产卵受精并形成存活后代的过程，证明在自然环境下，该四倍体群体群体能够自行繁殖传代。F₉—F₁₂染色体数目的稳定性和正常的性腺结构以及自然条件下的生殖传代行为，说明该异源四倍体鲫鲤已成为一个染色体数目为4n=200、遗传性状稳定的新型四倍体鱼群体，具备形成一个新的种群所需的关键因素。 2．人工诱导异源四倍体鲫鲤的雌核发育 异源四倍体鲫鲤能产生二倍体卵子，用UV处理镜鲤的精子，不进行染色体的加倍处理，能获得二倍体雌核发育鱼，通过冷休克对“受精卵”进行处理，仍然获得了二倍体雌核发育鱼。没有检测到雌核发育四倍体。这表明通过冷休克让二倍体卵子加倍成四倍体比较困难，但是经冷休克处理获得的雌核发育鱼的孵化率高于没有进行冷休克处理的。冷休克处理和未冷休克处理的雌核发育鱼均是二倍体（2n=100）的雌鱼，这证明了异源四倍体螂鲤是雌性的性别决定类型属于XXXX类型。二倍体雌核发育鱼的性成熟比普通的二倍体鲤、鱼即鱼的性成熟得晚，在一龄的雌核发育鱼没有发现达到性成熟的个体，只有到二龄时，才发行性成熟鱼。在第二年检测发现3070的雌核发育鱼是可育的，具有正常的卵巢，能产生成熟的卵子。在第三年可育的雌核发育鱼的比例有所增加。在外形上与湘螂FZ相似，在繁殖特性上，二倍体雌核发育鱼也与湘螂FZ相似，能产生二倍体卵子，其与红螂后代的染色体数为3n=150。3.人工诱导异源四倍体卿鲤的雄核发育 在2002年，将金鱼的在卵巢液保存下，用UV失活卵子的遗传物质。经UV处理的卵子与异源四倍体螂鲤的二倍体精子“受精”，诱导产生雄核发育鱼。观察发育过程发现，经照射过的卵子损伤比较大，到神经胚期，就大部分死亡，雄核发育鱼的孵化率极低，不到1%0，共得到8尾雄核发育仔鱼，鱼苗的死亡率很高，目前仅剩下两尾存活的雄核发育后代。目前还没有做进一步的染色体鉴定。2003年用失活的墨龙的卵子与四被体鱼的二倍体精子“受精”，获得了82尾雄核发育实验鱼。雄核发育的研究尚属起步阶段，有待进一步探索更好的诱导条件和改进诱导方法。更多还原

【Abstract】 The formation of the population of the allotetraploid hybrids of red crucian carp and common carp is the first case in the world which both the female and the male of allotetraploids are fertile, they are the good materials for studying the biological characteristic of tetraploid animals, this paper reports the hereditary characters of allotetraploids including the stability of hereditary gynogenesis and androgenesis.I.The chromosome number and gonadal structure of F₉-F₁2 a 11otetrapIo i d hybr i dsIn this paper, the chromosome number of of F₉-F₁2 of hybridis of Red crucian Carassius auratus （） × common carp Cryprinus carpio （） were examined by means of kidney cell-PHA culture. The results show that F9-F₁2 allotetraploida still possessed 200 chromosomes, with the karyotype formula 44m+68sm+44st+44t, which were the same as the data indicated in F₃-F₈ allotetraploids. The females and males of F₉-F₁2 possessed, respectively, the normal ovaries and testes that were able to produce the normal dipoid ova and sperm, respectively. With ultramicrotomy ,we observed the structure of the ovary and testis of allotetraploids, the mature testis are composed of lots of seminiferous tubules, which filled with spermatogoniunu spermatid and sperm, the diploid sperm consists of the head and the tail , the head is full of the dense chromatin, the central structure of tail is axial fiber with the "9+2" microtubules. There are lots of microvilli in the stage IVand V oocyte of the mature ovary, which stick into the zona pellucida, we concluded the function of the microvilli is exchanging the substance and singal. There aretwo yolksexist in the stage IVand Voocyte in two forms, one is the intravesicle yolk, the other is the extravesicle yolk. In nature conditions, without the injection of the extraneous hormone, the females and males of F₉-F₁2 could mate each other to produce new next generation of tetraploids. With the stable genetic tetraploidy and the fertility in the nature environments, this allotetraploid population possessed the key factors to form a new species with 200 chromosomes.2. The gynogenesis of allotetraploid hybridsWithout the treatment to double the chromosome number, the diploid eggs produced by the females of tetraploid hybrids of red crucian carpX common carp were able to develop to living diploid fish after subjecting to activation of the UV-irradiated sperm. With the cold shock, the diploid eggs also developed to diploid gynogens after the activation of the UV-irradiated sperm, no tetraploid gynogens being found, suggesting that it is difficult to double the chromosome from the diploid to tetraploid by means of cold shock treatment. The hatching rate of the gynogens with the cold shock was higher than that of the untreated gynogens. All the gyngens with and without cold shock were examined to be diploid females with 100 chromosomes, providing the evidence that the females of the tetraploid population are homogamety （xxx）. The mature females were only found among the two-year old gynogens, not being found among the one-year old gynogens, indicating that the ovary in the gynogens developed slowlier than that in the normal diploids such as the red crucian carp. Thirty percent of the gynogens were fertile which possessed the normal ovary and produced normal mature eggs. The appearance of the gynogenetic fish was similar to that of F2 hybrids, the diploidgynogens were able to produce diploid eggs that were proved by the presence of the triploids with 150 chromosomes among the offspring produced by mating the diploid females of the gynogens with the diploid males of the red crucian carp. As the control group, the diploid eggs fertilized with the haploid sperm of the scatter scale carp developed to triploid hybrids. The possible sex-determining types of the tetraploid population were also discussed in the present study. 3. The androgenesis of a Ilotetraploid hybridsIn 2002, the goldfish’ s female nucleus were inacted by using UV rays to irradiate the eggs, the diploid s更多还原