雌核发育彭泽鲫子代及双亲组织同工酶的比较研究

采用7．5％(ω)聚丙烯酰胺垂直板电泳技术研究了丰产鲫及其母本彭泽鲫和父本尖鳍鲤的心、肝、肾、脑4种组织的苹果酸酶(ME)、超氧化物歧化酶(SOD)和酯酶(EST)同工酶表型以及血清蛋白表型．结果表明：丰产鲫的血清蛋白和3种组织同工酶的电泳图谱均与母本彭泽鲫相同而与父本尖鳍鲤显著不同．说明丰产鲫是彭泽鲫卵子在尖鳍鲤精子激发下的雌核发育后代，在血清蛋白和同工酶表型的水平上，异源父本对子代成体的表型无影响。     

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.     

不同精子激发彭泽鲫卵子产生的子代及其双亲同工酶的比较

用彭泽鲫和尖鳍鲤的精子分别与彭泽鲫的卵子授精,产生2种子代PP和PS.采用聚丙烯酰胺凝胶电泳技术对子代及父母本3种组织(心脏、肾脏、肝脏)的4种同工酶(LDH,EST,SOD,MDH)以及肌肉蛋白进行了比较分析,结果表明:两种子代的同工酶表型几乎完全一致,且在肝脏LDH、肾脏EST和SOD以及肌肉蛋白表型上与母本彭泽鲫存在差异,说明精子来源的不同对子代成体的同工酶和肌肉蛋白表型没有影响.暗示在正常情况下,无论与同源精子还是异源精子授精,彭泽鲫都行雌核发育生殖方式.而子代与母本存在差异的事实又意味着彭泽鲫的雌核发育与传统意义上的单性型雌核发育鱼类不同,可能属于非典型的雌核发育.     

基于DNA含量的复合鲫倍性分析

通过对复合鲫血细胞DNA含量的测定,探讨了其倍性和产生机制等方面的问题.结果显示,复合鲫的DNA含量明显多于母本种彭泽鲫的DNA含量,且所测14尾中多数个体的DNA含量超过或接近四倍体的DNA含量,少数个体的DNA含量介于三倍体和四倍体之间.推测多数复合鲫个体为复合四倍体异育彭泽鲫,其染色体组包含母本彭泽鲫的全套染色体和父本单倍染色体组所产生的子代,少数个体可能为非整倍体.     

人工四倍体鲤鲫成熟卵受精过程的扫描电子显微镜观察

以人工四倍体鲤鲫卵子和普通红鲤精子为材料进行人工受精，在受精后的不同时间以扫描电子显微镜观察卵子对精子的应答反应、受精孔形态、数量与大小以及受精过程等超微结构特征. 结果表明，四倍体卵子动物极的卵壳上仅有一个受精孔,但受精孔区域有5—6个似小山丘状的嵴状结构，而鲤却完全没有这种结构. 研究证实：四倍体鲤鲫成熟卵属单受精孔、单精受精类型，其特异的嵴状形态结构可作为区分普通鲤的标记表型.     

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。     

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．     

异精雌核发育淇鲫胚胎发育过程中同工酶分析

分别用同源和异源精子激发的雌核发育淇鲫的12个发育时期的胚胎为材料,采用聚丙烯酰胺凝胶垂直平板电泳技术,对其5种同工酶(EST、LDH、MDH、ME和POD)的表达情况进行了比较分析.结果表明:在胚胎发育过程中,同源精子组和异源精子组胚胎的ME和EST同工酶表达无差异,LDH,MDH和POD同工酶表达存在显著差异.本文认为雌核发育淇鲫存在"异精生物学效应".     

异育彭泽鲫及其双亲消化酶活性的比较研究

对异育彭泽鲫及其双亲肝胰脏和前、中、后肠消化酶活性进行比较研究．结果表明:异育彭泽鲫肝胰脏及前、中、后肠的蛋白酶活性分别为367.69、101.16、297.06、25.97g/g#8226;min，均介于其双亲之间；异育彭泽鲫肝胰脏及前、中、后肠的淀粉酶活性分别为300.60、425.45、272.58、312.28g/g#8226;min，均大于双亲；异育彭泽鲫肝胰脏及前、中、后肠的脂肪酶活性分别为1.76、2.42、1.23、1.04g/g#8226;min，前肠和中肠脂肪酶活性大于双亲，后肠和肝胰脏脂肪酶活性介于双亲之间，且三种消化酶活性都比母本高.差异性分析结果表明，其与母本之间的差异显著低于其与父本的差异.研究结果符合雌核发育子代表型与母本一致的特性，并显示异源精子对雌核发育子代肝胰脏及肠管道消化酶活性有一定影响，为异育彭泽鲫的生长效应提供了一定的证据.     

异育淇鲫及其双亲的过氧化物酶和α-淀粉酶同工酶的比较研究

采用聚丙烯酰胺垂直平板电泳,对异育淇鲫及其母本淇鲫和父本兴国红鲤的6种组织(心脏、肝脏、肾脏、脑、晶状体和骨骼肌)中的过氧化物酶和α淀粉酶同工酶进行比较研究.结果显示:异育淇鲫同工酶的电泳图谱与母本淇鲫相同,与父本兴国红鲤显著不同.因而认为异育淇鲫是雌核发育的产物,父本遗传物质对子代无影响.     

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.     

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.     

滇池高背鲫鱼早期胚胎发育期间苹果酸脱氢酶(MDH)同工酶的分析

采用聚丙烯酰胺凝胶圆盘电泳技术，对滇池高背鲫鱼的成熟卵和胚胎不同发育时期的苹果酸脱氢酶（ＭＤＨ）同工酶的表达情况进行了比较分析，结果如下：１、在从未受精卵到肝形成期的整个发育过程中，ＭＤＨ持续地表达了五条酶带，即ＭＤＨ１－３、及ＭＤＨ７－８。始终持续表达的酶带，在发育过程中可能执行的是最基本的代谢功能，与细胞分化无关；２、随着胚胎发育的进展，在第Ｉ组胚胎，从肌肉效应期到血液循环期还出现了三条新的酶带，即ＭＤＨ－４，５，６。这可能反映了滇池高背鲫鱼种群内存在着ＭＤＨ基因和表达上的多态性     

滇池高背鲫同工酶发育遗传学研究

本研究采用聚丙烯酰胺凝胶圆盘电泳技术，对滇池高背鲫的成熟卵和胚胎之不同发育时期的醇脱氢酶（ＡＤＨ）、谷氨酸脱氢酶（ＧＤＨ）和碱性磷酸酶（ＡＬＰ）同工酶的表达进行了比较分析．结果表明：在从未受精卵到肝形成期的整个发育过程中，ＡＤＨ和ＧＤＨ两种同工酶均未检测出酶活性；ＡＬＰ在肝形成期以前也一直未表达，在肝形成期表达了一条酶带且有较高活性．ＡＬＰ的出现与滇池高背鲫肝组织的分化以及功能的建立之间表现出了高度的同步性，表明此酶带的出现与新个体生命活动加强，尤其与因摄食活动开始后消化系统的肝、肠、胃等的结构与功能的分化有关，是胚胎细胞核中相应基因开始表达的结果．此研究可为滇池高背鲫种群的生化遗传结构分析提供基础资料．