Comparative and Evolutionary Analysis of the Cytochrome b Sequences in Cyprinids with Different Ploidy Levels Derived from Crosses

The mitochondrial cyt b genes in the allotetraploid and triploid crucian carp as well as triploid common carp were isolated and completely sequenced. Their DNA sequences were compared with those derived from the cyt b genes of the red crucian carp, Japanese crucian carp, and common carp with MEGA 1.0 software. Phylogenetic analysis revealed the sister relationships between allotetraploid and diploid red crucian carp, between the triploid crucian carp and diploid Japanese crucian carp, and between triploid common carp and diploid common carp. Our results indicated the cyt b genes in the allotetraploid, triploid crucian carp, and triploid common carp were maternally inherited. Through maternal inheritance, the cyt b gene in the F11 tetraploid displayed extremely high similarity to that in the female parent red crucian carp after 11 generations (from F1 to F11 hybrids). Since the establishment of the new tetraploid stocks has great significance in analyzing evolutionary theory of vertebrate and in improving aquaculture industry, analysis of the cyt b gene and the elucidation of the variation of the cyt b gene DNA in different cyprinids prove that cyt b is a useful genetic marker to monitor the variations in the progeny of the crosses.     

从ATPase8-6基因研究杂交多倍体鱼线粒体母性遗传

异源四倍体鲫鲤是世界上首例人工培育的两性可育并形成群体的且能自然繁殖的四倍体鱼。本文采用质粒克隆测序法测定了红鲫、异源四倍体鲫鲤、三倍体湘云鲫和三倍体湘云鲤的ATPase8和ATPase6基因全序列 ,结合鲤鱼、日本白鲫和斑马鱼的同源序列 ,对不同倍性水平鲤科鱼类的ATPase8和ATPase6基因进行了比较 ,分析了碱基组成、变异情况以及核苷酸和氨基酸序列差异。红鲫、鲤鱼、异源四倍体鲫鲤、日本白鲫、三倍体湘云鲫和三倍体湘云鲤之间的序列差异为 0 0 % - 1 3 4 % ,它们与外群斑马鱼之间的序列差异为 2 7 9% -31 0 %。用MEGA软件中的MP法、ME法、NJ法和UPGMA法构建分子系统树 ,得到了相似的拓扑结构。结果分析表明 ,人工杂交多倍体异源四倍体鲫鲤、三倍体湘云鲫和三倍体湘云鲤在线粒体ATPase8和ATPase6基因上具有严格的母性遗传特征。值得注意的是 ,异源四倍体鲫鲤经过 1 1代的繁育后 ,与其原始母本红鲫仍然保持了非常高的同源性 ,说明了新的异源四倍体基因库在线粒体ATPase8和ATPase6基因上拥有稳定的遗传特性。对不同倍性鲤科鱼类线粒体ATPase8和ATPase6基因的研究表明 ,ATPase8和ATPase6基因是杂交鱼后代遗传变异研究的一个很好的分子标记     

Test systems for biomonitoring based on membrane-bound enzyme complexes. IV. Assessment of genotoxic effects in an Ames test system with metabolic activation by the microsomal mono-oxygenases from fish livers

The study of mutagenic effect of 2-aminoantracene and benz(alpha)pyrene on Salmonella triphimurium TA 100 in the Ames test-system in the presence of postmitochondrial fractions S-9 from carp liver with 3-methylcholantrene induced by microsomal oxidation system has been carried out. The metabolic activity and cytochrome P450 contence in carp liver microsomes have been shown to concede considerably those in rats liver. But these characteristics are sufficient for the use of fraction S-9 from carp liver for the study of genotoxic effect of these xenobiotics in the Ames test-system. Several regimes of storage of S-9 preparations from carp liver have been compared. S-9 preparations frozen immediately after isolation preserve their metabolic activity with respect to 2-aminoantracene and benz(alpha)pyrene well.     

Fertility of triploid hybrids of Prussian carp (<Emphasis Type="Italic">Carassius gibelio</Emphasis>) with common carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis> L.)

Fertility of backcross triploid hybrids containing one genome of Prussian carp and two genomes of common carp is investigated. The females of hybrids of Prussian carp and common carp (Prussian × common carp) are prolific and produce diploid gametes. Since males of such hybrids are sterile, their reproduction is realized by means of induced gynogenesis. Triploid progeny is obtained by backcrossing female Prussian × common carp with carp males. Among triploids obtained from hybrids F₁ and among hybrids of the first gynogenetic generation, there were no prolific specimens. However, in reproduction of diploid hybrids by means of gynogenesis during six generations, the female fertility in the backcross progeny is restored. From backcross triploid females (daughters of Prussian × common carp of the sixth gynogenetic generation), a viable triploid gynogenetic progeny and a tetraploid backcross (by carp) progeny are obtained. The obtained data may be considered as the experimental proof of the hypothesis of reticular speciation.     

Molecular cloning of the complement regulatory factor I isotypes from the common carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis>)

Factor I is a novel serine protease that regulates complement activation. Here we report the complete primary structure of two isotypic factor Is isolated from the common carp ( Cyprinus carpio), a pseudotetraploid teleost. A carp hepatopancreas cDNA library was screened using two RT-PCR-amplified cDNA fragments encoding part of the carp factor I-like serine protease domain. Two distinct cDNA clones, designated FI-A and FI-B, were isolated. Their deduced amino acid sequences share 75.2% identity with each other. FI-A has a typical factor I-like domain organization composed of two disulfide-linked polypeptides (H-chain and L-chain). On the other hand, FI-B contains a novel sequence of 115 amino acids inserted at the N-terminus of the H-chain. Genomic Southern hybridization suggests that FI-A and FI-B are encoded by distinct genes in the carp genome. Expression analysis by RT-PCR revealed that the major site of FI-A expression is the ovary, whereas FI-B expression is detected mainly in the hepatopancreas at a level higher than that of FI-A. The present data, taken together, suggest that carp have duplicated genes coding for factor I, and FI-B with the novel insertion plays a dominant role in the complement system. In addition, homology search of the fugu genome database using the carp FI-A and FI-B sequences identified a putative fugu factor I gene, which has an exon/intron organization different from that of the human orthologue.     

异源精子在银鲫雌核发育子代中的生物学效应

黑龙江省方正县双凤水库的两性型银鲫群体是三倍体雌核发育种群。异源精子不仅能刺激银鲫卵雌核发育,而且还能影响雌核发育子代的某些性状,如对于子代的生长、性比、体色和肝脏LDH同工酶等都产生了影响。为区别于原有术语“雌核发育gynogenesis”,我们把这种表现了异源精子生物学效应的雌核发育称之为“异精雌核发育allogynogenesis”,发育的子代称之为“异育银鲫”。异育银鲫已以其明显的生长优势在生产上显示了优良的经济性状。       

Some observations upon the relationship of microhaematocrit values to haemoglobin concentrations and erythrocyte numbers in the carp Cyprinus carpio L. and brook trout Salvelinus fontinalis (Mitchill)*

Simultaneous determinations of microhaematocrit, erythrocyte numbers and haemoglobin concentrations have been obtained for samples of carp and trout of both sexes acclimated to various temperature regimes, and varying seasonal and nutritional states. Because haematological parameters have been used as indices of physiological condition in fishes correlational and multivariant regression analyses were carried out in an effort to determine whether valid correlations existed between the readily-determined parameter, microhaematocrit, and the other 2 variables. Results obtained indicate that microhaematocrit can be used as a general index of haematological status in the carp but not the brook trout.     

金鱼ABC转运蛋白基因家族成员鉴定及分析

腺苷三磷酸结合盒转运蛋白(ATP-binding cassette transporter,ABC transporter)基因家族在原核生物和真核生物中广泛存在,该家族蛋白能够利用ATP裂解产生的能量将多种底物转运到膜上,参与多种生物过程,如营养摄入、细胞解毒、脂质稳态、信号转导、病毒防御以及抗原呈递等。目前,鱼类中,只在斑马鱼、斑点叉尾鮰和鲤鱼等少数鱼类中对该基因家族进行了系统的研究,关于金鱼ABC转运蛋白基因家族的详细分析,未见报道。本研究中,我们利用三代结合二代测序技术构建的金鱼转录组参考基因集数据,鉴定出55个ABC转运蛋白基因,通过系统进化分析将它们分为8个亚家族(A^H)。即金鱼ABC转运蛋白基因是由10个ABCA、14个ABCB、13个ABCC、5个ABCD、1个ABCE、4个ABCF、7个ABCG和1个ABCH组成。同时,我们将金鱼与斑马鱼、斑点叉尾鮰和鲤鱼等物种ABC转运蛋白基因家族成员的数目进行比较分析,推测硬骨鱼类特异的第3次全基因复制(3R-WGD)和谱系特异的第4次全基因组复制(4R-WGD)对金鱼该基因家族成员数目的影响。本研究结果为金鱼ABC转运蛋白基因功能的研究提供了理论依据。     

Phylogenetic and Evolutionary Analyses of the Frizzled Gene Family in Common Carp (Cyprinus carpio) Provide Insights into Gene Expansion from Whole-Genome Duplications

In humans, the frizzled (FZD) gene family encodes 10 homologous proteins that commonly localize to the plasma membrane. Besides being associated with three main signaling pathways for cell development, most FZDs have different physiological effects and are major determinants in the development process of vertebrates and. Here, we identified and annotated the FZD genes in the whole-genome of common carp (Cyprinus carpio), a teleost fish, and determined their phylogenetic relationships to FZDs in other vertebrates. Our analyses revealed extensive gene duplications in the common carp that have led to the 26 FZD genes that we detected in the common carp genome. All 26 FZD genes were assigned orthology to the 10 FZD genes of on-land vertebrates, with none of genes being specific to the fish lineage. We postulated that the expansion of the FZD gene family in common carp was the result of an additional whole genome duplication event and that the FZD gene family in other teleosts has been lost in their evolution history with the reason that the functions of genes are redundant and conservation. Through the expression profiling of FZD genes in common carp, we speculate that the ancestral gene was likely capable of performing all functions and was expressed broadly, while some descendant duplicate genes only performed partial functions and were specifically expressed at certain stages of development.     

人工诱导雌核发育日本白鲫

分别用遗传失活的散鳞镜鲤(Cyprinus carpio L.)、团头鲂(Megalobrama amblycephala)精子诱导日本白鲫(Carassius cuvieri)进行雌核发育.未经冷休克处理,用UV照射过的镜鲤的精子(其最佳UV照射剂量为4 200 mJ/cm2)与日本白鲫卵子受精获得(32.4±3.3)%雌核发育单倍体和(0.7±0.3)%杂交二倍体,而照射过的团头鲂精子(其最佳UV照射剂量为3 600mJ/cm2)与日本白鲫卵子受精得到了(33.8±1.4)%雌核发育单倍体和(0.5-±0.3)%杂交二倍体.日本白鲫卵子分别经灭活的镜鲤、团头鲂的精子激活(精子经各自最佳UV剂量处理),施以冷休克处理(受精后2min,0-4℃,40min),其孵化率分别为(27.8±2.1)%和(29.4±3.3)%,发育到摄食期,其正常的雌核发育二倍体鱼苗分别为(15.7±3.4)%和(23.6±4.1)%,在镜鲤实验组中,其正常的雌核发育二倍体鱼苗占孵化鱼苗总数的56%,这比团头鲂实验组中正常雌核发育二倍体鱼苗的比率(达到80%)低,结果表明诱导日本白鲫雌核发育,与母本遗传关系远的团头鲂精子较镜鲤的更有效.从染色体数目、外形特征和性腺结构等方面证实雌核发育后代的生物学特性.雌核发育日本白鲫全为雌性,这表明其雌性是同配XX型.雌核发育日本白鲫在生产上将有着潜在的价值,如比普通日本白鲫长得更快,抗病能力强等.所有的雌核发育日本白鲫的染色体数目都是100,而所有的团头鲂与日本白鲫的杂交后代都是三倍体(3n=124),新三倍体鱼在鱼类养殖和理论研究中将存在潜在的价值.     

Hereditary effects of artificial selection of sperm for the endurance trait in the carp Cyprinus carpio and the frog Rana temporaria

Studies have been made of the effect of artificial selection of parental sperm by maximum motility time (maximum tolerance) on the body length in carp larva and heat resistance level of muscle tissue in tadpoles. Sperm selection increases mean values of these parameters due to the increase in the progeny of the relative number of long-sized individuals and individuals with higher heat resistance of muscle fibers.     

Primary-structural and evolutionary analyses of the growth-hormone gene from grass carp (Ctenopharyngodon idellus).

The growth-hormone (GH) gene of grass carp, one of the fastest-growing species of farmed fish, was isolated and the DNA sequenced. Only one GH gene is found in this species. This gene, which is 2.5 kb in length, has five exons and four introns, in common with all of the mammalian and the recently published common-carp GH genes. In the course of vertebrate evolution, the total lengths of the intron and the non-coding region of exon 5 of the GH gene have been shortened by 40-70%, whereas the encoding exons of the gene have been slightly increased. The more closely related species exhibit the closest sequence similarity in their GH genes. For example, the similarity of the exons is 84.1-93.2% between grass carp and common carp (within the same family of Syprinedae), 43.5-82.1% between grass carp and rainbow trout (in different orders of Teleostei) and 45.8-58.6% between grass carp and rat (in different grades of Vertebrata). In addition, similar DNA domains, such as thyroid-hormone-receptor-complex-binding site and cell-type-specific cis elements involved in regulation of expression of rat and human GH genes, have been localized in the corresponding regions of the grass-carp GH gene.     

The immunological status of Cyprinus carpio L. in experimental mycotoxicosis

Some humoral factors of nonspecific resistance have been determined in carp yearlings in 28 days after intraperitoneal administration of fungi culture filtrate (I), conidia (II), micellar extract (III) of the fungus Aspergillus fumigatus Fres., physiological solution (control). The toxicogenic and immunogenic effect of the fungus has been shown in I and II variants of the experiment respectively. The injection of fungus micella did not have any influence on the studied factors. The account of specific and quantitative composition of zooparasites in experiments and under control has been carried out; no truly differences have been revealed.     

低温对鲤鱼的5个基因在不同组织中表达量的影响

本研究应用实时荧光定量 PCR技术对抑制性消减杂交所得到的鲤鱼不同温度下差异表达的5个基因做进一步的研究.结果表明: 水温由16℃降至4℃(骤降点)的过程中,在鲤鱼的肝、肠组织中,LKE-25基因的表达量呈下降趋势,而LKE-62基因则呈上升趋势;在肾脏组织中,LKE-48-1、 LKE-48-2和LKE-59基因的表达量呈上升趋势,且随着4℃维持时间的延长,其表达量逐渐下降.LKE-25和LKE-59基因在4℃同一尾鱼的心脏、脑、肝、脾、肾、肠组织中的表达量上有很大差异,其中LKE-25基因在心、LKE-59基因在肾组织中的表达量最高 .本实验所研究的5个基因在不同温度、不同组织表达量的显著差异,证明了这些基因与水温相关且有组织特异性 [动物学报 54(3):460-466,2008].     

人工雌核发育鲢的遗传多样性及异源遗传物质整入的RAPD分析

运用RAPD技术对连续二代人工雌核发育鲢的遗传多样性及异源遗传物质的整入进行了分析 ,结果表明 :一代雌核发育鲢 ,个体间遗传相似度为 0 94 5— 0 995 6 ,多样性指数为 0 175 ;二代雌核发育鲢 ,个体间遗传相似度为0 96 15— 1 0 0 ,平均为 0 985 2 ,多样性指数为 0 0 6 2。研究揭示经过连续二代人工雌核发育后 ,其遗传多样性明显减少 ,种质进一步纯化。通过对雌核发育鲢二代、亲本鲢和雄鲤的RAPD扩增比较 ,发现雌核发育鲢含有少数与父本相同的特异DNA扩增带 ,而亲本鲢没有 ,在基因水平上表明雌核发育鲢整入了雄鲤的遗传物质     

Multiple inserts of gene of interest and selectable marker gene are co-integrated and stably transmitted as a single genetic locus in transgenic soybean plants

Particle bombardment has been used for soybean transformation for more than 20 yr, but the integration and segregation of transgene inserts in the soybean genome have not been clearly documented. Over the past 5 yr, we processed several hundred transgenic events. In each experiment, the expression cassettes of the gene of interest (GOI) and hygromycin selectable marker gene (SMG) were co-bombarded into soybean at a 1:1 molecular ratio. More than 75% of hygromycin-resistant events also carried the GOI. Molecular analysis of transgenic plants revealed that most events carried multiple inserts of the GOI and the SMG. The GOI and the SMG were linked in selfed T1 and T2 progeny. Segregation analysis of progeny indicated that, in over 98% of the transgenic events, the multiple inserts of the GOI were integrated into the same genetic locus resulting in a 3:1 segregation ratio. Furthermore, the multiple inserts of the GOI are transmitted into succeeding generations, and no recombinants were found. These data indicate that in soybean plants, co-bombarded genes are preferentially integrated and stably segregated as a single genetic locus.