摘要
通过红鲫(Carassius auratus red var.,♀)与鲤鱼(Cyprinus carpioL.,♂)远缘杂培育出两性可育的异源四倍体鲫鲤群体(AT)。AT能稳定产生二倍体精子和卵子,对其产生的二倍体卵子进行雌核发育研究,建立了一个能稳定产生二倍体卵子的雌核发育二倍体鲫鲤克隆体系(G1～G5)。利用雌核发育二倍体鲫鲤克隆体系产生的二倍体卵子与四倍体鲫鲤产生的二倍体精子杂交获得了改良四倍体鲫鲤G×AT。重要的是,在改良四倍体鲫鲤群体(G1×AT)中选育出2%的高背型个体。高背型G1×AT自交,后代分化形成3种二倍体鱼：高背型红鲫、高背型双尾金鱼和高背型青灰色鲤鱼。本文主要研究内容包括：①雌核发育二倍体鲫鲤克隆体系的建立及其遗传机制研究；②对人工双尾金鱼的细胞和分子生物学特性进行深入分析,来探讨金鱼的起源与进化问题；③高背型鲫鱼和高背型青灰色鲤鱼的生物学特征及其应用。
具体研究内容如下：
1.在已获得雌核发育二倍体鲫鲤第三代G3的基础上,制备出G4和G5。建立了一个能稳定产生二倍体卵子的雌核发育二倍体鲫鲤克隆体系(G1～G5)。为进一步研究二倍体鱼产生二倍体卵子的机制,我们对G5早期性腺进行体外组织培育。在早期性腺生长增殖中,发现大量生殖细胞发生融合的现象。而在对照组早期草鱼和鲤鱼性腺的体外培育中,至今未发现生殖细胞融合的现象。这对揭示二倍体杂交鱼产生二倍体配子的机制具有重要的价值。
2.对高背型人工双尾金鱼(AG)的外形及细胞生物学特征进行研究。结果表明,AG与普通金鱼(NG)的可量性状和可数性状基本一致；AG与原始母本RCC的一些外形特征更为相似,而与原始父本CC的外形特征差异较大。从外形特征上可以初步判断金鱼与鲫鱼的亲缘关系比较近。流式细胞仪测定结果表明AG是一种二倍体鱼。染色体检测表明AG染色体数为2n=100,染色体核型公式为22m+34sm+22st+22t。通过对AG性腺的显微结构和超微结构观察,发现它们具有正常的精巢和卵巢结构,两性个体一年性成熟,分别能产生成熟的单倍体精子和卵子。
3.在AG分子遗传特性研究中,首先对NG、AG、RCC和CC的Sox基因进行检测。AG与RCC的Sox基因扩增条带一致,同为-200、-600和～1900bp的扩增带,而在NG中只存在-200和～600bp的条带,CC中只存在～200和～900bp的扩增带。另外,对这四种鱼的5S rDNA进行PCR扩增,结果表明NG与AG具有相同的扩增带,它们以具有独特的168bp条带而异于RCC和CC。同时在对5S rDNA进行染色体定位研究中,发现NG、AG和RCC的原位杂交结果一致,它们都具有两个较强的杂交信号,而在CC染色体中没有检测到相关信号。
对AG线粒体DNA全长进行测序。AG粒体基因组全序列长为16579bp,其结构和基因排列与其他鲤科鱼一致。对线粒体全序列进行比对,AG与原始亲本RCC和CC的相似比分别为99.5%和89.4%,符合线粒体母性遗传规律；AG与NG的相似比高达99.4%,表明两者可能具有相同的起源。
从鲤鱼和鳙鱼的微卫星引物中筛选出7对引物对NG、AG、RCC和CC群体进行微卫星检测。结果表明,在AG群体中各等位基因数为1-8个,大小在140-300bp,群体呈现了高度的遗传多态性,遗传异质性较大,表明基因组来源复杂,基因高度杂合。
4.对高背型鲫鱼相关生物学特征进行研究,结果表明它们是-种二倍体鱼,两性可育,一年性成熟；它们在外形上具有体背高、尾柄短、头部小的优良特性。用雌性高背型鲫鱼与雄性改良四倍体鲫鲤交配,获得了改良三倍体鲫鱼。这种改良三倍体鲫鱼不仅在外形上继承了母本高背特征,而且具有生长速度快、不育、营养成分含量高等特征。
5.对高背型青灰色鲤鱼(ICC)及其自交后代的外形、倍性、性腺发育以及Sox基因进行研究。结果表明ICC与普通鲤鱼(CC)相比,ICC在外形上具有显著的高背特征。ICC是一种二倍体鱼,两性可育,年性成熟。值得注意的是Sox基因检测结果表明ICC是一种杂交个体,具有与AT一致的扩增带(-200、-600、-900和～1900bp),而CC为～200和～900bp的扩增带。
上述研究结果说明经雌核发育效应改良的远缘杂交后代能分化形成了不同类型的新品种鱼,它们在外形和遗传特性方面都发生了重大变化。对三种不同种类高背型鱼的形成及生物学特征进行研究,在生物进化和遗传育种中具有重要意义。而人工双尾金鱼的形成为研究金鱼的起源与进化提供了直接的证据。
A bisexual fertile allotetraploid (AT) population was obtained by crossing red crucian carp (Carassius auratus red var.,♀,2n=100) with common carp(Cyprinus carpio L., (?),2n=100). Gynogenesis was researched on the diploid eggs produced by AT, and a gynogenetic diploid hybrid clone line was established. The diploid eggs produced by gynogenetic diploid hybrid clone line were fertilized with the diploid sperm of AT to produce a new type of tetraploid fish (G×AT,4n=200). Importantly,2% of G1×AT with the obviously high body height to body length were found in G1×AT population. Self-mating of the high-body G1×AT produced three kinds of bisexual fertile diploid fish:high-body red crucian carp, high-body fork-like-tails goldfish and high-body gray common carp. The main contents of this paper included:①Establishment and genetic mechanism of the diploid gynogenetic hybrid clonal line.②In-depth analysis the cell and molecular biological characteristics of AG to provide insight into the evolutionary origin of goldfish.③Application and biological characterization of high-body crucian carp and high-body gray common carp. The major results were presented as follows:
1. Based on the formation of the third gynogenetic generation (G3), G4 and G5were obtained by gynogenesis. Thus, a diploid gynogenetic hybrid clonal line (G1-G5) of red crucian carp X common carp was established. For further study of the mechanism of diploid fish produce diploid eggs, we cultivated the early-stage gonadal cells of G5 in vitro. During the gonad proliferation stage, we found the phenomenon of germ cell fusion. While in the control group, grass carp and common carp gonads, there was no phenomenon of cell fusion. These results were of great value to reveal the mechanism why diploid hybrids produce the diploid gametes.
2. The morphological characteristics of artificial goldfish (AG) were observed. The results showed that AG and the natural goldfish (NG) with the similar traits in morphological. Compared with original parents RCC and CC, AG showed more similar characteristics with RCC, but it was great different from CC. It can determine the genetic relationship between goldfish and crucian carp from the morphological traits. AG possessed 100 chromosomes with the karyotype 22m+34sm+22st+22t, and the DNA content was same with diploid RCC. Both ovary and testis gonads of AG were able to produce normal mature gametes after one year culture.
3. To detect the molecular genetic characteristics of AG, the Sox genes of NG, AG, RCC and CC were detected firstly. AG and RCC amplified the same bands consistent with the-200,-600 and-1900bp, in NG there were only-200 and-600bp bands, and in CC there were only-200 and-900bp bands. PCR amplification of 5S rDNA results showed that AG and NG with the same amplified band, and both of them had a unique band (168bp) different from RCC and CC.5S rDNA fluorescence in situ hybridization results showed that AG has two strong hybridizing signals as RCC and NG, while in CC there was no signal detected.
The complete sequence of mitochondrial DNA of AG was 16579bp. Compeared with original parents RCC and CC, the similar rate were 99.5% and 89.4%, respectively, in line with the maternal inheritance of mitochondria. However, AG had the high similarity rate with NG (99.4%), indicating that they may have the same origin.
The number of alleles varied from one to eight in AG, and the size of the alleles ranged from 140-300bp. The amplification patterns of 7 pairs of microsatellite primers showed that AG had a higher degree of genetic polymorphism, a greater genetic heterogeneity, indicating that the source of genome was complexity and the gene was highly hybrid.
4. The biological characteristics of high-body crucian carp were studied, the results revealed that high-body crucian carp was a diploid and bisexual fertile fish, possessed many improved features such as smaller head, higher body and shorter caudal. An improved triploid was produced by crossing improved tetraploids with improved diploids. The improved triploids showed some features such as high-body, fast growth, infertility, and high nutrient contents.
5. The phenotypes, chromosome numbers, gonadal structure and Sox gene of ICC were observed. The results revealed that ICC was a diploid and bisexual fertile fish, and showed the significant features of the high-back compared with CC. It was worth noting that the Sox gene amplification results showed that ICC was a hybrid, which had the same amplified bands with AT (-200,-600,-900 and-1900bp), while CC had-200 and-900bp bands.
These results indicated that to improve the distant crossing hybrids by gynogenetic, the offspring can differentiate into different types of new varieties of fish, which appearance and genetic characteristics had major changes. Such of various improved fishes plays an important role in biological evolution and fish genetic breeding. Furthermore the formation of AG provided the direct evidence for the evolutionary origin and diversification of goldfish.
引文
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