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题名: (左鱼右丹)亚科三种鱼类的单性发育及纯系建立
作者: 贾方钧
答辩日期: 2001
导师: 吴清江
专业: 遗传学
授予单位: 中国科学院水生生物研究所
授予地点: 中国科学院水生生物研究所
学位: 博士
关键词: 斑马鱼 ; 稀有(左鱼右句)鲫 ; 雄核发育 ; 雌核发育 ; 性别遗传 ; 胚胎融合
摘要: 本论文的研究分别以(左鱼右丹)亚科(Danioninae)的三种小型鱼类为材料,探讨了实现鱼类单性发育的不同途径。研究内容分为三个部分。第一章,通过连续两代的雌核发育,即第一代的有丝分裂雌核发育和第二代的极体雌核发育,结合表型及分子水平上的遗传标记的筛选鉴别,建立了斑马鱼(Danio rerio)的三个纯(近交)系。品系遗传标记的建立为物种生物学特性的分析提供了有力的工具。所确定的遗传标记,通过紫外线促使卵子遗传失活的方法,实现了斑马鱼的核发育。通过对两个品系间的杂交个体的自发雌核发育子代的遗传标记的分析,反映出自发雌核发育的个体继承了母本的杂合状态,因此推测自发雌核发育可能与天然雌核发育有内在的相关性。通过对一个家系的连续六个世代的雌核发育的跟踪观察,发现每个世代中都有少数的雄性个体存在;而在雄核发育研究获得的子代中也发现有两种性别的个体存在,说明斑马鱼所性别遗传不符合单对性染色体决定的传统模式,作者因此提出了斑马鱼常染色体决定性别的假想模型。第二章:本章的研究旨在解决当缺乏直观的种内遗传标记进获得可靠的人工单性发育后代的问题。在初步分析了稀有(左鱼右句)鲫(Gobiocypris ratus)的染色体组型特征之后,进行了稀有(左鱼右句)鲫的人工单性发育的研究。诱导人工雌核发育使用了鲤鱼等远缘种的精子,确保避免受到由于精子遗传失活不彻底可能在子代中产生杂种。经过对热休克参数的比较分析,得出了诱导极体雌核发育的最佳条件为受精后2min, 40 ℃持续处理2min,二倍体仔鱼的相对存活率为12.2%;诱导有丝分裂雌核发育的最佳条件为受精后17分钟、40 ℃持续2分钟,休克处理的二倍体仔鱼相对存活率为3.4%。研究稀有(左鱼右句)鲫人工雄核发育使用了经地~(60)Co-γ处理的异种卵子,采用的几种卵子均被证明不能与稀有(左鱼右句)鲫产生可存活的二倍体杂种。实验结果表明,大斑马鱼的卵质能够为稀有(左鱼右句)鲫雄核的发育提供必要的环境,在经过电激诱发二倍化的处理之后获得了正常存活的雄核发育二倍体。稀有(左鱼右句)鲫人工单性发育的子代个体的性别组成表明,稀有(左鱼右句)鲫的性别遗传接近于经典的XY型性别遗传模式。第三章:本章的研究采用大斑马鱼为材料,探讨了由电激诱发胚胎融合实现染色体组人工操作的方法,并对大斑马鱼的生物学特性进行了初步观察。直流电激处理2细胞初期的受精卵,能有效地达到胚胎细胞融合实现染色体组合加倍的效果。经过对融合条件的比较分析,结果显示电脉冲持续时间为40μs, 在0.25mol/L甘露醇液中对单倍体胚胎施加电场强度360V/cm的单个直流电脉冲,胚胎融合率达到84.9%,囊胚期存活率为58.9%,二倍化率为55.2%,雌核发育仔鱼存活率在6.2%左右。用同样的条件诱导正常二倍体胚胎的融合以获得四倍体的尝试也获得了初步的成功。实验结果也显示交流电场对2细胞胚胎的融合没有效果。对大斑马鱼生物学特性的观察显示大斑马鱼同稀有(左鱼右句)鲫和斑马鱼等已经得到较多的鱼类实验动物一样具有性成熟快、产卵频次高、胚胎易于观察等优点,可以成为供选择的鱼类实验动物之一。
英文摘要: Three species of Danioninae fishes were employed to investigate the methods of production of homozygous fishes, and three main parts were included in the present dissertation. Chapter I: With two consecutive gynogenetic generations, namely the first mitotic gynogenetic generation and the second meiotic gynogenetic generation associated with the screening of genetic markers at phenotype and molecular levels, three inbreed strains of zebrafish (Danio rerio) were constructed. The determination of strain-specific markers provided the powerful tools to explore the biological features of the species. Based on the screened markers, androgenetic zebrafish were produced with UV irradiated eggs. The analysis to the genetic markers in spontaneous gynogenetic diploid originated from the crossing between two strains revealed the phenomena that spontaneous gynogenetic diploid inherited the maternal heterozygosity, which suggests the correlation between spontaneous diploid gynogenesis and natural gynogenesis. To retrieve a family propagated for six consecutive gynogenetic generations, a minority of male individuals were occurred in each generation, while both male and female individuals were occurred in an androgenetic family, which inflicted with the classic model of sex determination by single pair of sex chromosomes. Consequently, the author suggested a hypothesis that the sex determination in zebrafish might be regulated by autochromoses. Chapter II: The goal in this study referred to produce reliable homozygous progeny when plain intraspecies markers were not available. The karyotype of rare minnow (Gobiocypris rarus) was analyzed in this chapter at first, and then, the artificial unisexual reproduction of G. rarus was investigated. To avoid the possibility that hybrids were produced due to incomplete deactivation of sperms, sperms of distant species such as common carp were employed. Through the evaluating of parameter for heat shocking experiments, the optimum was determined as heat shocking at 2min after fertilization, 40 ℃ for 2min with the meiotic gynogenesis, in which the relative survival rate of diploid fries was 12.2%; with the mitotic gynogenesis, the optimum was at 17min after fertilization, 40 ℃ for 2min, and the relative survival rate of diploid fries was 3.4%. ~(60)Co-γ irradiated eggs from different distant species were employed for the production of androgenetic rare minnow, which were confirmed that the viable hybrids would not be produced between them and rare minnow. The results showed that, the ooplasm of giant zebrafish (Danio aequipinnatus) would serve the development of male nucleus of rare minnow, and viable androgenetic fries of rare minnow were produced after the induction of diploidization with electric pulse. The sexual composition of progeny produced through artificial unisexual reproduction suggested the model of sex determination in rare minnow would be likely matched the classic XY model of sex determination. Chapter III; Giant zebrafish was employed to investigate the method of embryonic fusion induced with electric pulse, and the biological characteristics of giant zebrafish were observed as well. The chromosome set could be doubled if fertilized eggs at 2-cell stage were exposed to direct electric pulse. Through several batches of experiments, the results showed that haploid embryos immersed in the medium of 0.25M mannitol and exposed to a single direct electric pulse with the field intensity at 360V/cm and duration for 40μs would produce 6.2% normal fries, with which the rate of embryonic fusion should reach at 84.9%, survival at blastula stage reach at 58.9% and diploidization reach at 55.2%. Following the above protocol, fusing normal diploid embryos also produced some tetraploid individuals. The effects of alternating electric fields on embryonic fusion were excluded in this study. Compared with zebrafish and rare minnow, the giant zebrafish exhibited the similar quality as laboratory animal such as quick sex maturation, high spawning and ready to observe during embryonic development, which serve it an optional laboratory animal for scientific purpose.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.ihb.ac.cn/handle/342005/12608
Appears in Collections:中科院水生所知识产出(2009年前)_学位论文

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Recommended Citation:
(左鱼右丹)亚科三种鱼类的单性发育及纯系建立.贾方钧[d].中国科学院水生生物研究所,2001.20-25
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