复合四倍体彭泽鲫雌核发育生殖方式的 胚胎同工酶证据

复合四倍体彭泽鲫是在尖鳍鲤精子刺激的彭泽鲫雌核发育子代中发现的少数特殊个体．本文分别用母本种彭泽鲫和父本种尖鳍鲤的精子与复合四倍体彭泽鲫的卵子受精，得到两种胚胎FP和FS．采用聚丙烯酰胺凝胶垂直板电泳技术对两种胚胎10个时期的5种同工酶（LDH、MDH、ME、SOD和EST）进行比较分析．结果表明:两种胚胎的5种同工酶表型在谱带上完全一致，不同来源的精子对胚胎的同工酶表型没有影响．表明复合四倍体彭泽鲫可能继承了母本种的雌核发育生殖方式．     

复合四倍体彭泽鲫胚胎发育的研究

分别用复合四倍体彭泽鲫的母本种彭泽鲫和父本种尖鳍鲤的精子与复合四倍体彭泽鲫的卵子授精,在Nikon体视显微镜下对它们的胚胎发育过程进行连续观察.描述了23个发育时期的主要形态特征,观察了不同水温条件对胚胎发育速率的影响,比较了复合四倍体彭泽鲫与异育彭泽鲫胚胎发育过程的异同.发现二者在受精卵的大小、受精后皮质颗粒、肌节对数、眼晶体形成时间、积温等方面都存在差异,复合四倍体彭泽鲫比异育彭泽鲫的胚胎发育历时要少.在上述研究的基础上,发现来自母本种和父本种的两种不同精子与复合四倍体彭泽鲫的卵子授精,得到的胚胎在发育过程中未表现出明显差异,暗示复合四倍体彭泽鲫的卵子在应答母本种和父本种精子时表现出相同的生殖方式,即雌核发育生殖方式.本研究的结果和结论与在人工复合四倍体异育银鲫中观察到的现象存在显著差异.     

不同倍性鲤科鱼Cyclin B基因内含子克隆与进化分析

运用PCR扩增、克隆、测序等技术,分别获得三倍体鲫鲂、四倍体鲫鲂、五倍体鲫鲂和团头鲂、草鱼、鲢鱼、鳙鱼等鲤科鱼Cyclin B基因部分DNA序列.结合异源四倍体鲫鲤及其原始亲本红鲫和鲤鱼cyclin B基因对应DNA序列,对不同倍性鲤科鱼类cyclin B基因DNA片段序列进行比较分析.结果表明:由相同引物扩增的染色体数为48的鲂、草、鲢、鳙鱼,只扩增出1条DNA片段,片段长度分别为750 bp,950 bp,720 bp和720 bp,而染色体数目加倍的红鲫、鲤鱼、异源四倍体鲫鲤、异源四倍体鲫鲂,扩增出2条DNA片段(1200 bp和900 bp),三倍体和五倍体鲫鲂扩增出3条DNA片段(1200 bp,900 bp和750 bp),每个DNA片段均含Cyclin B基因第2,3内含子和第2,3,4外显子.序列分析表明,四倍体鲫鲤、三倍体鲫鲂、四倍体鲫鲂和五倍体鲫鲂与其母本1200 bp片段同源性分别为99.5%,98.9%,99.5%和88.7%,与母本900 bp片段同源性分别为97.5%,94.6%,94.2%和89.9%,说明四倍体鲫鲤与多倍体鲫鲂1200 bp和900 bpCyclin B基因片段与母本红鲫同源性高,在进化上具有偏母性遗传特性.而三倍体和五倍体鲫鲂的750 bp Cyclin B基因片段与父本同源性分别高达98.6%和98.2%,说明其来自父本团头鲂.在两性可育的异源四倍体鲫鲤和异源四倍体鲫鲂中存在各自父本Cyclin B基因片段序列消除现象.此外,用Cyclin B基因内含子3序列构建不同倍性鲤科鱼的系统进化树,结果表明,由Cyclin B基因内含子序列构建的系统进化树可以正确反映亲缘关系较近的鲤亚科属间鱼类系统进化关系,而不适合亲缘关系较远的亚科间杂交鱼类系统进化分析.     

不同倍性鲤科鱼CyclinB基因内含子克隆与进化分析

运用PCR扩增、克隆、测序等技术,分别获得三倍体鲫鲂、四倍体鲫鲂、五倍体鲫鲂和团头鲂、草鱼、鲢鱼、鳙鱼等鲤科鱼CyclinB基因部分DNA序列．结合异源四倍体鲫鲤及其原始亲本红鲫和鲤鱼CyclinB基因对应DNA序列,对不同倍性鲤科鱼类CyclinB基因DNA片段序列进行比较分析．结果表明：由相同引物扩增的染色体数为48的鲂、草、鲢、鳙鱼,只扩增出1条DNA片段,片段长度分别为750bp,950bp,720bp和720bp,而染色体数目加倍的红鲫、鲤鱼、异源四倍体鲫鲤、异源四倍体鲫鲂,扩增出2条DNA片段（1200bp和900bp）,三倍体和五倍体鲫鲂扩增出3条DNA片段（1200bp,900bp和750bp）,每个DNA片段均含CyclinB基因第2,3内含子和第2,3,4外显子．序列分析表明,四倍体鲫鲤、三倍体鲫鲂、四倍体鲫鲂和五倍体鲫鲂与其母本1200bp片段同源性分别为99．5％,98．9％,99．5％和88．7％,与母本900bp片段同源性分别为97．5％,94．6％,94．2％和89．99／6,说明四倍体鲫鲤与多倍体鲫鲂1200bp和900bpCyclinB基因片段与母本红鲫同源性高,在进化上具有偏母性遗传特性．而三倍体和五倍体鲫鲂的750bpCyclinB基因片段-9父本同源性分别高达98．6％和98．2％,说明其来自父本团头鲂．在两性可育的异源四倍体鲫鲤和异源四倍体鲫鲂中存在各自父本CyclinB基因片段序列消除现象．此外,用CyclinB基因内含子3序列构建不同倍性鲤科鱼的系统进化树,结果表明,由CyclinB基因内含子序列构建的系统进化树可以正确反映亲缘关系较近的鲤亚科属间鱼类系统进化关系,而不适合亲缘关系较远的亚科间杂交鱼类系统进化分析．     

异源四倍体鲫鲤群体的形成及四倍体化在脊椎动物进化中的作用

对雌雄两性能育并形成群体的异源四倍体鲫鲤的染色体数目和组型,DNA含量,红细胞大小,生殖腺和配子,胚胎发育,形成机理,外形等方面进行了较全面描述。四倍体鲫鲤经过九代（F3-F11）的四倍体性的繁殖,已形成了一个数目庞大的遗传性状稳定的群体。该四倍体群体在染色体数目、生殖、外形特征等方面都与它们的原始父母本-二倍体湘江野鲤和红鲫有本质的差别。在遗传特性、稳定传代、生育隔离等方面,异源四倍体鲫鲤群体为形成一个新的四倍体新种奠定了基础。新的四倍体鱼群体的形成对脊椎动物的进化理论和它们在生产上的应用都具有重要的意义。     

人工四倍体鲤鲫的产生

在人工诱导的复合三倍体鲤鱼成熟个体中有极个别个体产出的卵既保留了原有的３套染色体,又能与一套外来精核融合产生四倍体。１９９７年人工产生的８０００多尾鲤鲫四位体鱼经细胞遗传学研究分析鉴定,结果所有的个体都有４套染色体组,染色体数目在１８５￣２１０之间,众数为２００,四位体比率为１００％,这表明人工复合三倍体鲤鱼的生殖方式十分独特,绝大部分个体的成熟卵可借助于外来精子的流动进行自然雌核发育;而有极个别     

洞庭青鲫和彭泽鲫DNA提取及其RAPD分析

DNA样品检测结果表明:彭泽鲫基因组DNA的平均产率为735,洞庭青鲫基因组DNA的平均产率为447.1;从80个随机引物中筛选出5个引物对彭泽鲫和洞庭青鲫两个群体进行了RAPD分析.结果发现彭泽鲫群体多态位百分率为57.5%,群体内遗传相似度和遗传距离分别为0.850和0.150;洞庭青鲫群体多态位点百分率为56.65%,遗传相似度和遗传距离分别为0.927和0.073;两群体间相似率和遗传距离分别为0.811和0.189.这表明洞庭青鲫群体具有较低的遗传多样性,彭泽鲫群体内的遗传多样性相对较高,但洞庭青鲫与彭泽鲫两群体间存在遗传差异.     

利用ATPase基因研究杂交多倍体鲫鲂线粒体母性遗传

三倍体鲫鲂、四倍体鲫鲂和五倍体鲫鲂是通过红鲫旱×团头鲂舍亚科间远缘杂交形成的．采用PCR产物直接测序法,测定了5尾三倍体鲫鲂、6尾四倍体鲫鲂和6尾五倍体鲫鲂及其1尾父本团头鲂的线粒体DNA ATPase8和ATPase6基因的全序列,并与所获得的红鲫和外群斑马鱼的同源序列进行比较,同时分析了其碱基组成、变异情况以及核苷酸和氨基酸序列差异．红鲫、团头鲂、三倍体鲫鲂、四倍体鲫鲂和五倍体鲫鲂之间的序列分歧率为0．0％-21．6％,它们与外群斑马鱼之间的序列分歧率为27．0％-28．2％．用MEGA3．1软件中的Maximum parsimony（MP）,Minimum evolution（ME）,Neighbor joining（NJ）和UPGMA程序构建的分子系统树具有相似的拓扑结构．结果表明,人工杂交三倍体鲫鲂、四倍体鲫鲂和五倍体鲫鲂在线粒体ATPase8和ATPase6基因上具有严格的母性遗传特征．研究证明ATPase8和ATPase6基因是杂交多倍体鲫鲂遗传变异研究的一个很好的分子标记．     

不同倍性鲫鲂高迁移率族蛋白1基因的克隆和原核表达

用RACE PCR技术,分别获得三倍体鲫鲂、四倍体鲫鲂、红鲫和团头鲂的高迁移率族蛋白1基因(HMG1)mRNA全长序列,其开放阅读框包含579nt,翻译成193个氨基酸.不同倍性鲫鲂HMG1的cDNA和氨基酸序列比较分析表明:在cDNA水平上,四倍体鲫鲂与母本红鲫的同源性(99%)高于同父本团头鲂的同源性(97%);三倍体鲫鲂与父母本同源性(95%)低于亲本之间的同源性(98%);在氨基酸水平上,四倍体鲫鲂与父母本的同源性(100%)高于三倍体鲫鲂与亲本的同源性(97%).结果表明:远源物种间的杂交对两性不育的三倍体鲫鲂HMG1基因造成了一定的冲击,分子遗传效应表现为三倍体鲫鲂HMG1基因位点发生了变异;四倍体鲫鲂HMG1氨基酸序列与亲本的完全一致性,克服了等位基因的杂交不亲和性,为两性可育的异源四倍体鲫鲂遗传稳定奠定了基础.生物信息学分析表明:HMG1蛋白二级结构具有8个螺旋和3个转角,HMG1蛋白三级结构于N-端具有两个DNA结合基序,C-端具有一长为23个重复D或E的氨基酸尾.这种结构决定了HMG1能够与核DNA发生"蛋白-DNA"的相互作用,参与多种核内生物学功能的完成.另外,以HMG1氨基酸序列构建了鱼类、两栖类、爬行类、鸟类和哺乳类的进化树,结果提示HMG1是一种古老的蛋白质,并在物种演化中具有保守性;首次构建了鱼类HMG1原核表达载体,外源的鱼类HMG1基因编码蛋白在原核细胞中得到了表达,这为下一步HMG1蛋白的制备和生物学功能、尤其与DNA转座子相互作用的研究提供了条件,将助于了解物种间的杂交形成异源多倍体的机制.     

彭泽鲫繁殖特性的研究

用水平式淀粉凝胶电脉法,分析了5组47尾实验鱼的肌肉和肝脏的4种同工酶,研究了彭泽鲫的繁殖特性,结果表明：彭泽鲫（♀）与彭泽鲫（♂）的子代、彭泽鲫（♀）与鲤鱼（♂）的子代的同工酶电泳图谱与母本完全相同,不含有来自于父本的遗传物质,可以认为彭泽鲫是行雌核发育的种群,子代应是与母本的遗传组成完全相同的克隆。     

应用RAPD和同工酶遗传标记鉴别彭泽鲫中的克隆

应用随机扩增多态性DNA(RAPD)和同工酶检测技术检测分析了18尾彭泽鲫，从样品鳍条提取基因组DNA，水平式淀粉凝胶电泳法检测同工酶，结果显示，肝脏GPI、肌肉GPI和肝脏EST3种同工酶均有变异出现，3种同工酶组合后可将实验鱼划分为遗传组成各不相同的8种克隆，6种引物PCR扩增的RAPD电泳图谱与同工酶的划分结果一致。     

Cloning and evolutionary analysis of cyclin B gene introns in cyprinids with different ploidy levels

Partial fragments of the cyclin B gene from triploid, tetraploid, and pentaploid hybrids of red crucian carp × blunt snout bream, blunt snout bream, grass carp, silver carp, and bighead carp were amplified. One DNA fragment was amplified from the blunt snout bream, grass carp, silver carp, and bighead carp (750, 950, 720, and 720 bp, respectively). Two fragments (1200 and 900 bp) were amplified from the red crucian carp, common carp, and allotetraploids. The triploid and pentaploid hybrids yielded three DNA fragments (1200, 900, and 750 bp). The 1200 bp fragment of the allotetraploid crucian carp, triploid, tetraploid, pentaploid hybrids of red crucian carp × blunt snout bream shared 99.5%, 98.9%, 99.5%, and 88.7% homology, respectively, with the maternal DNA. The 900 bp fragment shared 97.5%, 94.6%, 94.2%, and 89.9% homology, respectively. Our results suggest that inheritance is maternally dominated. Furthermore, we observed preferential elimination of the paternal sequences in the allotetraploid hybrids. Based on these sequence analyses we constructed a phylogenetic tree to explain the relationships among the different ploidy levels.     

Comparative analysis and evolutionary significance of the HMG1 gene in crucian carp,blunt snout bream,and their polyploid progeny

The full-length mRNA of the high mobility group protein 1 coding gene (HMG1) was obtained by RACE-PCR from red crucian carp (Carassius auratus red var.),blunt snout bream (Megalobrama amblycephala),and their triploid and tetraploid progeny.The sequence contained an open reading frame of 579 nucleotides coding for 193 amino acids.The nucleotide identity of HMG1 was higher between the tetraploid hybrid and the maternal red crucian carp (99%) than between the tetraploid hybrid and the paternal blunt snout bream (97%).The nucleotide identity between the triploid hybrids and each parent (95%) was lower than that between the parents (98%).The protein identity between the tetraploid hybrid and each parent (100%) was higher than that between the triploid hybrid and each parent (97%).Our results suggest that interspecific hybridization generates a shock to the HMG1 gene in triploid hybrids,causing divergence of nucleotides.The HMG1 protein of the tetraploid hybrids was consistent with that of its parents,which reduced the barrier of cross incompatibility between alleles,providing the basis for the bisexual fertile tetraploid hybrids forming a new polyploid species in nature.The secondary and tertiary structures of the HMG1 protein contain eight helices,three switches,two DNA-binding domains in the N-terminus,and a long acidic tail in the C-terminus.Together,these data suggest that the HMG1 protein plays a role of protein-DNA interactions,facilitating various DNA-dependent activities in the nucleus.We also investigated the phylogeny of fish,amphibian,reptilian,bird,and mammalian HMG1 proteins.Our results suggest that HMG1 is an ancestral protein that has been highly conserved.These data provide clues as to how interspecific hybridization may form polyploid hybrids.     

银鲫人工杂交试验中雄核发育现象的RAPD实验证据

以随机扩增多态性ＤＮＡ（ＲＡＰＤ）技术为手段，对生野鲫，雄性异育银鲫及其杂交ＦＩ代（选择性状接近父本的５％Ｆ１代），父本回交Ｆ１你进行了遗传多态分析，从１５０个随机序列引物中筛选得到９种引物，并用它们可得到３６个用作遗传标记的ＲＡＰＤ多态性条带，其中的２２条父本显性条带均遗传子代，而１４条母本性条带则均未被遗传子代，对两亲本和子代中的ＲＡＰＤ标记条带的分布情况及相似系数分析，以及聚类分析结果均支持     

137 Cs-γ 辐照对实验红鲫 AFLP 多态性的影响

摘要:目的 观察137 Cs-γ 辐照对实验红鲫 AFLP 的变化,探讨不同剂量137 Cs-γ 辐照对实验红鲫 DNA 多态性的影响。 方法 根据前期实验结果,将实验红鲫 C1HD 系随机分为 5 组,10 尾 / 组,即空白对照组、1 / 16 LD50 、1 / 8 LD50 、1 / 4 LD50 、1 / 2 LD504 个辐照组,设置 1. 94 Gy、3. 88 Gy、7. 76 Gy、15. 53 Gy4 个辐照剂量组和一个空白对照组,用生物辐照仪对实验红鲫进行一次性辐照。 提取辐照前和辐照后的实验红鲫血液 DNA,经 AFLP-PCR 扩增,并进行多态性信息含量( PIC) 、基因杂合度( H) 、香农信息指数( I) 分析。 结果 实验红鲫在137 Cs-γ 辐照处理前的 PIC、H 和 I分别为 45. 20% 、0. 19 和 0. 28,经137 Cs-γ 辐 照 处 理 后 分 别 为 72. 27% 、0. 28 和 0. 41,且 遗 传 学 指 数 升 高。 结 论经137 Cs-γ 辐照处理后,实验红鲫的 AFLP 多态性信息含量增加,基因杂合度升高。 AFLP 标记实验红鲫 C1HD 系可用于监测核辐射污染。     

Maternal inheritance in polyploid fish inferred from mitochondrial ATPase genes analysis

The sequences of the ATPase8/6 genes for the triploid, tetraploid and pentaploid hybrids as well as for their male parent blunt snout bream were determined. In order to examine mitochondrial maternal inheritance, the sequences were subjected to a comparative sequence analysis with the homologous sequences of red crucian carp, their female parent, and zebrafish as the outgroup. Base composition and variation as well as the divergences based on nucleotide sequences and deduced amino acid sequences were calculated. Phylogenetic trees were also constructed with maximum parsimony (MP), minimum evolution (ME), neighbor joining (NJ) and the unweighted pair group method with arithmetic mean (UPGMA) algorithms in MEGA 3.1. The results showed that most nucleotide substitutions occurred at the third codon position of the two genes and thus represented synonymous mutations. The nucleotide sequence divergences of the ATPase8/6 genes ranged from 0.0% to 21.6% among ingroup samples (three types of polyploids and their parents), and 27.0–28.2% between their ingroup and the outgroup samples. All the polyploids were considerably closer in sequence relationship to the female parent red crucian carp (0.0–3.3%) compared to their male parent blunt snout bream (21.0–21.6%). The phylogenetic trees also showed a similar result. In conclusion, the mitochondrial ATPase8/6 genes of artificial polyploid fish stringently indicated maternal inheritance. Our results also suggested that the ATPase8/6 genes are valuable genetic markers to track genealogies and variations in the progenies of the hybrids.     

Analysis of intron sequence variability of the conservative HMG-box of Sox9 genes in allotetraploids and their original parents

The Sox genes of allotetraploids and their original maternal red crucian carp (Carassius caassius red var.) and original paternal common carp (Cyprinus carpio L.) were detected by PCR with the designed primers based on the conserved HMG-box sequence in different species. Sequencing of Sox genes indicated that two Sox9 genes (Atsox9a and Atsox9b) existed in allotetraploids, while only one Sox9 gene existed in red crucian carp (Rcsox9a) and common carp (Ccsox9b) . All of the four Sox9 genes contained an intron in the HMG-box. with the sizes of 413 bp, 703 bp, 401 bp and 714 bp, respectively. Moreover, the introns obeyed the rule of "GT-AG" . A high similarity was observed between introns of Atsox9a and Rcsox9a (94.4%), Atsox9b and Ccsox9b (97.8%). Interestingly, the deduced amino acid sequences of their corresponding exons all shared 100% identity. Thus, introns of the HMG-domain of Sox9s in allotetraploids and their original parents have not only the length polymorphism but also intron variability. Our results provide significant molecular evidence for the origin and evolution of allotetraploids.