Molecular mechanisms of gynogenetic clonal reproduction in polyploid crucian carp

多倍体鲫鱼雌核发育克隆繁殖的分子机制

基本信息

批准号：
11660289
负责人：
FUJITANI Hideo
金额：
$ 2.18万
依托单位：
Azabu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11660289/
关键词：
Carassius auratus langsdorfi gynogenesis polyploidy cDNA subtraction D-loop clonal reproduction DNA markers wee1 フナ DNA多型マーカー 3倍体 RDA法倍数性ディファレンシャルディスプレイ(DD)法

项目摘要

This three-year project was to study the molecular mechanisms underlying gynogenesis practiced by a population, or subspecies, of Japanese crucian carp, namely, Carassius auratus langsdorfi. This extremely rare and interesting vertebtare(fish) has been known to produce unreduced eggs with its genomic constitution exactly identical to that of somatic cells by means of 1. skipping the first meiotic division and 2. upon insemination, keeping the membrane of incoming sperm nucleus undamaged, thus prohibiting the male genome from participating in embryo development. Such a type of clonal reprodction as this one is remarkable in terms of both basic developmental biology and developing an artificial way of mass production of clonal offspring.In this study, we examined a sizable collection of C.a.langsdorfi (both the bisexsual diploid and all-female tri- or tetraploid) and several other C.a. subspecies, including the goldfish, from many parts of Japan for their mitochondrial D-loop sequences, DNA markers which we found specific/polymorphic, electrophoretograms of RAPD-PCR with carefully selected primers, and we almsot surely clarified the maternal origin of the genomes of the gynogenetic populations. In the course of this investigation, a strong link between the triploid genome and genomes of both diploid C.a. and goldfish was also found.In search for genes that are related to gynogenetic mechanisms, we chose two approaches : close examination of wee1 gene, an important cell-cycle regulating gene, and mutual subtraction of cDNAs from overies of both diploid and triploid fish. For the former, we conducted a thorough characterization of wee1 gene and its product. For the latter, four genes were isolated which were reasonably expected to be related to the gynogenetic phenomena and they were analyzed for the base sequences and patterns of expression.

这个为期三年的项目旨在研究日本鲫鱼种群或亚种（即 Carassius auratus langsdorfi）进行雌核发育的分子机制。这种极其罕见和有趣的脊椎动物（鱼）已知可以产生未减数的卵子，其基因组构成与体细胞的基因组构成完全相同，方法是：1.跳过第一次减数分裂，2.在授精时，保留传入精子核的膜未受损，从而阻止雄性基因组参与胚胎发育。这种类型的克隆繁殖在基础发育生物学和开发大规模克隆后代的人工方法方面都是引人注目的。在这项研究中，我们检查了相当大的 C.a.langsdorfi 收藏品（包括双性二倍体和全染色体）。雌性三倍体或四倍体）和其他几个 C.a.来自日本许多地区的包括金鱼在内的亚种的线粒体 D 环序列、我们发现的特异性/多态性 DNA 标记、使用精心挑选的引物进行 RAPD-PCR 的电泳图，我们几乎肯定澄清了金鱼基因组的母本起源雌核发育种群。在这项研究的过程中，三倍体基因组和两个二倍体 C.a. 的基因组之间存在紧密的联系。为了寻找与雌核发育机制相关的基因，我们选择了两种方法：仔细检查重要的细胞周期调节基因wee1基因，以及从二倍体和三倍体鱼的cDNA中相互扣除。对于前者，我们对 wee1 基因及其产物进行了彻底的表征。对于后者，分离了四个基因，这些基因被合理地预期与雌核发育现象相关，并对它们的碱基序列和表达模式进行了分析。