【作者】 何伟国；

【导师】 刘筠； 刘少军；

【作者基本信息】 湖南师范大学 ， 发育生物学， 2012， 博士

【摘要】 多倍化是指基因组中额外一套或多套染色体的渗入现象,他在植物和动物中是一种流行的物种形成机制。导致多倍化的途径有多种,包括体细胞加倍、非减数配子的融合、多精受精或“三倍体桥”。现在普遍认为杂交在引发多倍化过程中起着重要的作用。多倍体物种在植物中相当常见,然而在动物中则相对较少。即便如此,多倍化现象在许多鱼类中却广泛存在着。并且,许多人工诱导的多倍体鱼已经用来制备不育的鱼类品种和促进生产。另外,鱼类中的多倍体还可以作为一种有用的模式系统用来检验基于植物研究得出的关于多倍化起源和结果的理论。在本研究中,通过远缘杂交和遗传选育的生物学方法与技术,将鲤科鱼类中的红鲫(Carassius auratus red var,, RCC♀$,2n=100)与翘嘴红鱼鲌(Erythroculter ilishaeformis Bleeker, TC♂2n=48)杂交在脊椎动物中首次获得了存活的二倍体鲫鲌(2nRT)、三倍体鲫鲌(3nRT)和四倍体鲫鲌(4nRT)杂交鱼。并且,对这些不同倍性水平杂交鱼的倍性、染色体数目、核型、DNA含量、外形特征、Sox基因、5S rDNA的结构组成与变异以及他们在染色体上的定位情况等生物学特征进行了系统研究,主要研究内容如下：1)通过远缘杂交技术将雌性红鲫与雄性翘嘴红鲌杂交获得了二倍体鲫鲌、三倍体鲫鲌和四倍体鲫鲌三种不同倍性水平的杂交鱼后代。并利用染色体计数、核型分析和血细胞平均DNA含量测定方法对这些杂交鱼后代及其亲本的倍性水平和染色体组成情况进行了鉴定。二倍体红鲫含有100条染色体,核型公式为22m+34sm+22st+22t:二倍体翘嘴红鲌含有48条染色体,核型公式为16m+26sm+6st.在杂交鱼后代中,二倍体鲫鲌的染色体数目为74,核型公式为19m+30sm+14st+11t,其中一套来自于红鲫,一套来自于翘嘴红鲌；三倍体鲫鲌的染色体数目为124,核型公式为30m+47sm+25st+22t,其中两套来自于红鲫,一套来自于翘嘴红鲌；四倍体鲫鲌的染色体数目为148,核型公式为38m+60sm+28st+22t,其中两套来自于红鲫,两套来自于翘嘴红鲌。另外,对不同倍性水平杂交鱼及其亲本的早期胚胎发育过程进行了系统观察,并对他们的受精率、孵化率和成活率进行了统计分析。2)对两年龄的三种不同倍性水平杂交鱼后代的可数性状和可比性状进行了系统分析。结果表明杂交鱼后代中许多性状均介于红鲫与翘嘴红鲌之间,体现出杂交特征。以相同时期的红鲫与翘嘴红鲌作为对照,对二倍体鲫鲌和三倍体鲫鲌的性腺显微结构和超微结构进行了检测,并在繁殖季节对二倍体鲫鲌和三倍体鲫鲌进行了人工催产繁殖,结果表明二倍体鲫鲌与三倍体鲫鲌均不可育。由于群体数量过少的缘故,未对四倍体鲫鲌性腺的显微结构和超微结构进行观察,也未对其进行人工催产,其育性有待进一步研究。3)参照Sox基因保守的HMG框序列,设计合成一对简并引物对不同倍性水平杂交鱼后代及其亲本的全基因组DNA进行了PCR扩增和测序分析,并通过对比不同倍性水平杂交鱼后代与他们亲本的同源性,对他们的分子遗传关系进行了系统分析。在红鲫中扩增出了215bp、617bp和1958bp三种大小的DNA条带；在翘嘴红鲌只扩增出了215bp大小的DNA条带；在二倍体鲫鲌中扩增出了215bp.628bp和1959bp三种大小的DNA条带；在三倍体鲫鲌中扩增出了215bp、616bp和1957bp三种大小的DNA条带；在四倍体鲫鲌中除了扩增出215bp、628bp和1957bp大小的三种DNA条带外,还衍生出了一条大小为918bp的DNA条带。4)对红鲫、翘嘴红鲌及其不同倍性水平杂交鱼后代的5S rDNA的结构与变异情况进行了系统分析。在红鲫中含有三种大小分别为203bp、340bp和477bp的5S rDNA结构单元,分别命名为结构单元Ⅰ、结构单元Ⅱ和结构单元Ⅲ；在翘嘴红鲌中含有两种大小分别为188bp和286bp的5S rDNA结构单元,分别命名为结构单元Ⅳ和结构单元Ⅴ。在杂交鱼后代中,二倍体鲫鲌从母本红鲫遗传获得了结构单元Ⅰ、结构单元Ⅱ和结构单元Ⅲ,从父本翘嘴红鲌部分遗传了结构单元Ⅳ；三倍体鲫鲌从红鲫只部分遗传了结构单元Ⅱ和结构单元Ⅲ,从翘嘴红鲌只遗传获得了结构单元Ⅳ；四倍体鲫鲌只从红鲫部分遗传获得了结构单元Ⅱ和结构单元Ⅲ,另外还衍生了一种新的5S rDNA序列,命名为结构单元Ⅰ-N。父本特异的5S rDNA结构单元Ⅴ在三种不同倍性水平杂交鱼后代中均未发现。5)通过荧光原位杂交(FISH)技术,以红鲫与翘嘴红鲌特异的5S rDNA片段(RCC-340bp, TC-188bp, TC-286bp)质粒为模板、PCR法扩增制备的5S rDNA荧光探针分别与红鲫和翘嘴红鲌及其不同倍性水平杂交鱼后代的染色体中期分裂相进行杂交,对红鲫和翘嘴红鲌及其不同倍性水平杂交鱼后代5S rDNA的染色体定位情况进行了研究分析。研究结果表明杂交和多倍化对杂交鱼后代基因组中5S rDNA结构单元位点产生了较大的影响,不仅有新5S rDNA结构单元位点的出现,也有位点的丢失。更多还原

【Abstract】 Polyploidization, the addition of an extra set (or sets) of chromosomes to the genome, is a predominant mechanism for speciation in plants and animals. Polyploidization can occur via somatic doubling, the fusion of unreduced gametes, and by means of a triploid bridge or polyspermy. The current prevailing opinion is that hybridization plays an important role in triggering polyploidization. Polyploid species are particularly frequent in the plant kingdom. However, while it is generally relatively rare in animals, polyploidy has occurred extensively, independently, and is often repeated in many groups of fish. Furthermore, artificially induced polyploidy has been used in aquaculture to produce sterility and to improve production. Polyploidy in fish represents a useful model system with which to test theories about the origin and consequences of polyploidy that have been derived from work on plants. Through distant crossing, diploid, triploid and tetraploid hybrids of red crucian carp (Carassius auratus red var., RCC♀, Cyprininae,2n=100)×topmouth culter (Erythroculter ilishaeformis Bleeker, TC♂,Cultrinae,2n=48) were successfully produced. This is the first report on the formation of these viable diploid, triploid and tetraploid hybrids by crossing different parents with a different chromosome number in vertebrates. In addition, studies of these different ploidy-level hybrid offspring on ploidy-level, chromosomal number, karyotype, mean DNA content, appearance, Sox genes, the organization and variation of5S rDNA and location of5S rDNA on the chromosomes were conducted, and the major contents are as follows:1) Through distant crossing, diploid, triploid and tetraploid hybrids of red crucian carp (RCC♀) x topmouth culter (TC♂) were successfully produced. The different ploidy-level and chromosome constitution of these hybrid offspring were determined with chromosome counting and detection of mean DNA content of blood cells. The distribution of chromosome number in RCC, TC,2nRT,3nRT and4nRT hybrids are as follows:For diploid RCC, the chromosomal metaphases had100chromosomes with the karyotype formula of22m+34sm+22st+22t; For diploid TC, the chromosomal metaphases possessed48chromosomes with the karyotype formula of16m+26sm+6st. In2nRT hybrid offspring of RCC♀±TC♂, the chromosomal metaphases had74chromosomes with the karyotype formula of19m+30sm+14st+11t, indicating diploid hybrids possessed74chromosomes with one set from RCC and one set from TC1In3nRT hybrid offspring of RCC♀×TC♂, the chromosomal metaphases had124chromosomes with the karyotype formula of30m+47sm+25st+22t, suggesting triploid hybrids harbored124chromosomes with two sets from RCC and one set from TC; In4nRT hybrid offspring of RCC♀×TC♂, the chromosomal metaphases had148chromosomes with the karyotype formula of38m+60sm+28st+22t, showing tetraploid hybrids had148chromosomes with two sets from RCC and two sets from TC. In addition, the earlier embryonic development of RCC, TC and their hybrid offsrping were detected, and the fertilization rate, hatching rate and adulthood rate were also involved.2) At2years of age, the measurable and countable traits in2nRT,3nRT and4nRT hybrids were examined, which showed the hybrid characteristics. Using RCC and TC as controls, the observation of microstructure and ultrastructure in2nRT and3nRT hybrids was conducted. An artificial propagation of2nRT and3nRT hybrids was also involved during the reproductive season, but neither egg nor sperm were able to be squeezed out from2nRT or3nRT hybrids. Results obtained here indicated that2nRT and3nRT hybrids were sterile. Due to the few number of4nRT hybrids, neither the detection of microstructure and ultrastructure nor an artificial propagation of4nRT hybrids was conducted. The confirmation of fertility in4nRT hybrids needs further study.3) Against the conserved HMG-box of Sox genes, one pair of degenerate primers were designed and synthesized to amplify the genomic DNA of RCC, TC and their different ploidy-level hybrid offspring. There were three DNA fragments of Sox genes (with the length of215bp,617bp and1958bp) in RCC, only one (with the length of215bp) in TC. In the hybrid offspring, there were three DNA fragments (with the length of215bp,628bp and1959bp) in2nRT hybrids, three DNA fragments (with the length of215bp,616bp and1957bp) in3nRT hybrids, four DNA fragments (with the length of215bp,628bp,918bp and1959bp) in4nRT hybrids. A novel918bp DNA fragment was observed in4nRT hybrids.4) The5S rDNA of the three different ploidy-level hybrids and their parents were sequenced and analyzed. There were three monomeric5S rDNA classes (designated class I:203bp; class II:340bp; and class III:477bp) in RCC and two monomeric5S rDNA classes (designated class IV:188bp, and class V:286bp) in TC. In the hybrid offspring,2nRT hybrids inherited three5S rDNA classes from their female parent (RCC) and only class IV from their male parent (TC).3nRT hybrids inherited class II and class III from their female parent (RCC) and class IV from their male parent (TC).4nRT hybrids gained class II and class III from their female parent (RCC), and generated a new5S rDNA sequence (designated class I-N). The specific paternal5S rDNA sequence of class V was not found in the hybrid offspring.5) Through fluorescence in situ hybridization (FISH), the fluorescence probes amplified from the parental-special5S rDNA fragments of RCC and TC (RCC-340bp, TC-188bp and TC-286bp) were hybridized to the mitotic metaphase chromosomes of RCC, TC,2nRT,3nRT and4nRT hybrids, respectively, and the location of5S rDNA loci on the chromosomes were also involved. The results obtained here indicated that hybridization and polyploidization had a huge impact on the5S rDNA loci, including the appearance of novel5S rDNA loci and lose of5S rDNA loci.更多还原